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1.
ACS Appl Mater Interfaces ; 10(48): 41056-41069, 2018 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-30387987

RESUMO

Intracellular activation of nanomaterials within cancer cells presents a powerful means to enhance anticancer specificity and efficacy. In light of upregulated lysosomal protease cathepsin-B (CathB) in many types of invasive cancer cells, herein, we exploit CathB-catalyzed biodegradation of acetylated rapeseed protein isolate (ARPI) to design polymer-drug nanocomplexes that can produce proapoptotic peptides in situ and synergize chemotherapy. ARPI forms nanocomplexes with chitosan (CS) and anticancer drug doxorubicin (DOX) [DOX-ARPI/CS nanoparticles (NPs)] by ionic self-assembly. The dual acidic pH- and CathB-responsive properties of the nanocomplexes and CathB-catalyzed biodegradation of ARPI enable efficient lysosomal escape and nuclei trafficking of released DOX, resulting in elevated cytotoxicity in CathB-overexpressing breast cancer cells. The ARPI-derived bioactive peptides exhibit synergistic anticancer effect with DOX by regulating pro- and antiapoptotic-relevant proteins ( p53, Bax, Bcl-2, pro-caspase-3) at mitochondria. In an orthotopic breast tumor model of CathB-overexpressing breast cancer, DOX-ARPI/CS NPs remarkably inhibit tumor growth, enhance tumor cell apoptosis and prolong host survival without eliciting any systemic toxicity. These results suggest that exploitation of multifunctional biomaterials to specifically produce anticancer agents inside cancer cells and trigger drug release to the subcellular target sites is a promising strategy for designing effective synergistic nanomedicines with minimal off-target toxicity.


Assuntos
Brassica rapa/química , Neoplasias da Mama , Catepsina B/biossíntese , Doxorrubicina , Portadores de Fármacos , Nanoestruturas , Proteínas de Neoplasias/metabolismo , Proteínas de Armazenamento de Sementes , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Feminino , Humanos , Células MCF-7 , Nanoestruturas/química , Nanoestruturas/uso terapêutico , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/farmacocinética , Proteínas de Armazenamento de Sementes/farmacologia
2.
Comp Biochem Physiol B Biochem Mol Biol ; 221-222: 18-28, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29649577

RESUMO

Cathepsin B is a lysosomal proteolytic enzyme that has been suggested to play a role in pathological processes of immune system. In this study, the full-length cDNA sequence of cathepsin B transcript in the giant river prawn Macrobrachium rosenbergii (MrCTSB) was obtained from 454 pyrosequencing of cDNAs from hepatopancreas and muscle. It was 1158 bp in length, containing an open reading frame (ORF) of 987 bp corresponding to 328 amino acids. The predicted molecular mass and pI of MrCTSB protein was 36.04 kDa and 4.73. The major characteristics of MrCTSB protein consisted of a propeptide of C1 peptidase family at the N-terminus and a cysteine protease (Pept_C1) domain at the C-terminus. The 3-dimentional structure of MrCTSB was constructed by computer-assisted homology modeling. The folding of MrCTSB was highly conserved to human CTSB structure and the modeled MrCTSB displayed characteristics of cysteine proteinases superfamily. The docking study was performed to investigate binding interactions between known inhibitors against MrCTSB. Known inhibitors were oriented in the groove of catalytic site cleft. They bound to subsites from S2, S1, S1', and S2', respectively, with key residues in each subsite. Challenge of juvenile prawns with Aeromonas hydrophila revealed that the MrCTSB transcript in hepatopancreas significantly increased at 60-96 h post injection (hpi). This suggested that MrCTSB may play roles in innate immunity of M. rosenbergii. Our results provide useful information for a more comprehensive study in immune-related functions of MrCTSB.


Assuntos
Aeromonas hydrophila , Proteínas de Artrópodes , Catepsina B , Regulação Enzimológica da Expressão Gênica , Palaemonidae , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , Catepsina B/biossíntese , Catepsina B/genética , Biologia Computacional , Palaemonidae/enzimologia , Palaemonidae/genética , Palaemonidae/microbiologia
3.
Cancer Res ; 78(10): 2524-2535, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29510992

RESUMO

CHIP/STUB1 ubiquitin ligase is a negative co-chaperone for HSP90/HSC70, and its expression is reduced or lost in several cancers, including breast cancer. Using an extensive and well-annotated breast cancer tissue collection, we identified the loss of nuclear but not cytoplasmic CHIP to predict more aggressive tumorigenesis and shorter patient survival, with loss of CHIP in two thirds of ErbB2+ and triple-negative breast cancers (TNBC) and in one third of ER+ breast cancers. Reduced CHIP expression was seen in breast cancer patient-derived xenograft tumors and in ErbB2+ and TNBC cell lines. Ectopic CHIP expression in ErbB2+ lines suppressed in vitro oncogenic traits and in vivo xenograft tumor growth. An unbiased screen for CHIP-regulated nuclear transcription factors identified many candidates whose DNA-binding activity was up- or downregulated by CHIP. We characterized myeloid zinc finger 1 (MZF1) as a CHIP target, given its recently identified role as a positive regulator of cathepsin B/L (CTSB/L)-mediated tumor cell invasion downstream of ErbB2. We show that CHIP negatively regulates CTSB/L expression in ErbB2+ and other breast cancer cell lines. CTSB inhibition abrogates invasion and matrix degradation in vitro and halts ErbB2+ breast cancer cell line xenograft growth. We conclude that loss of CHIP remodels the cellular transcriptome to unleash critical pro-oncogenic pathways, such as the matrix-degrading enzymes of the cathepsin family, whose components can provide new therapeutic opportunities in breast and other cancers with loss of CHIP expression.Significance: These findings reveal a novel targetable pathway of breast oncogenesis unleashed by the loss of tumor suppressor ubiquitin ligase CHIP/STUB1. Cancer Res; 78(10); 2524-35. ©2018 AACR.


Assuntos
Catepsina B/metabolismo , Catepsina L/metabolismo , Transformação Celular Neoplásica/genética , Neoplasias de Mama Triplo Negativas/patologia , Ubiquitina-Proteína Ligases/metabolismo , Catepsina B/biossíntese , Catepsina L/biossíntese , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Células MCF-7 , Receptor ErbB-2/metabolismo , Receptores Estrogênicos/metabolismo , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/mortalidade , Ubiquitina-Proteína Ligases/genética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Mol Reprod Dev ; 84(1): 67-75, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27862569

RESUMO

Growth factors synthesized by ovarian somatic cells affect cumulus cell expansion and oocyte maturation in vitro. Fibroblast growth factor 10 (FGF10), for example, is a known regulator of mammalian cumulus-oocyte complex maturation. In this study, we investigated the effects of 0, 5, 10, 50, and 100 ng/mL FGF10 (5F, 10F, 50F, and 100F, respectively) on in vitro cumulus cell expansion, oocyte maturation, and embryo development. The percentage of fully expanded cumulus cells at the oocyte's metaphase-II (MII) stage was significantly higher in the 10F-treated group than in the control. Transcript abundance of the cumulus cell expansion-related gene encoding hyaluronian synthase 2 (HAS2) in cumulus cells at oocyte germinal vesicle breakdown (GVBD) was significantly higher in the 10F- and 50F-treated groups compared to untreated controls, whereas the mRNA abundance of the protease cathepsin B (CTSB) at the oocyte MII stage was remarkably decreased in the 10F-treated group. The percentage of oocytes with normal spindles was greater in the 10F- and 50F-treated group at GVBD than in the other groups; the 5F-, 10F-, and 100F-treated groups were higher than the control; and the 50F-treated group was highest at MII. The abundance of GDF9 and BMP15 transcript at GVBD and BMP15 and CCNB1 transcripts at MII increased in the 10F-treated group. Cleavage rate, blastocyst formation rate, and total cell number were significantly higher in the 5F- to 50F-treated groups. These results demonstrate that FGF10 markedly improves cumulus cell expansion, oocyte maturation, and subsequent embryo development. Mol. Reprod. Dev. 84: 67-75, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Células do Cúmulo/metabolismo , Fator 10 de Crescimento de Fibroblastos/farmacologia , Oócitos/metabolismo , Animais , Proteína Morfogenética Óssea 15/biossíntese , Catepsina B/biossíntese , Células Cultivadas , Células do Cúmulo/citologia , Feminino , Fator 9 de Diferenciação de Crescimento/biossíntese , Hialuronan Sintases/biossíntese , Oócitos/citologia , Suínos
5.
J Mol Cell Cardiol ; 86: 32-41, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26163874

RESUMO

AIMS: Macrophage inflammation response is important in the pathogenesis of atherosclerosis. We investigated the role and mechanism of cellular repressor of E1A-stimulated genes (CREG) in regulating TNF-α induced inflammation response in macrophages and explore whether CREG might be a therapeutic target for atherosclerosis. METHOD AND RESULTS: Immunostaining and western blotting showed that expression of CREG was reduced in human atherosclerotic coronary artery. In vivo experiments demonstrated that supplementation of recombinant CREG protein to ApoE(-/-) mice fed with high fat diet alleviated aortic atherosclerosis development and inflammation. In vitro, macrophage from ApoE(-/-) mice fed with high fat diet had lower level of CREG compared to control mice fed with normal diet. Immunohistochemical staining and western blotting further confirmed that CREG inhibited inflammatory response of macrophages induced by TNF-α. Supplementation of exogenous recombinant CREG protein or CREG gene silencing showed that CREG promoted autophagy in TNF-α treated macrophages. The use of autophagy inhibitors, 3-methyladenine and bafilomycin A, identified that CREG attenuated TNF-α induced inflammation by activate autophagy. In addition, supplementation of exogenous CREG protein stimulated expression and maturity of cathepsin B and cathepsin L and induced lysosome formation, whereas CREG deficiency reduced lysosomal formation. CONCLUSION: CREG inhibits inflammation and promotes autophagy mediated by lysosome formation; it might be a potential therapeutic target in atherosclerosis.


Assuntos
Aterosclerose/genética , Inflamação/genética , Proteínas Repressoras/biossíntese , Fator de Necrose Tumoral alfa/administração & dosagem , Animais , Apolipoproteínas E/genética , Aterosclerose/patologia , Catepsina B/biossíntese , Catepsina L/biossíntese , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Lisossomos/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Proteínas Repressoras/genética , Fator de Necrose Tumoral alfa/metabolismo
6.
Biochim Biophys Acta ; 1851(10): 1304-1316, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26143381

RESUMO

During autophagy, autophagosomes fuse with lysosomes to degrade damaged organelles and misfolded proteins. Breakdown products are released into the cytosol and contribute to energy and metabolic building block supply, especially during starvation. Lipophagy has been defined as the autophagy-mediated degradation of lipid droplets (LDs) by lysosomal acid lipase. Adipose triglyceride lipase (ATGL) is the major enzyme catalyzing the initial step of lipolysis by hydrolyzing triglycerides (TGs) in cytosolic LDs. Consequently, most organs and cells, including macrophages, lacking ATGL accumulate TGs, resulting in reduced intracellular free fatty acid concentrations. Macrophages deficient in hormone-sensitive lipase (H0) lack TG accumulation albeit reduced in vitro TG hydrolase activity. We hypothesized that autophagy is activated in lipase-deficient macrophages to counteract their energy deficit. We therefore generated mice lacking both ATGL and HSL (A0H0). Macrophages from A0H0 mice showed 73% reduced neutral TG hydrolase activity, resulting in TG-rich LD accumulation. Increased expression of cathepsin B, accumulation of LC3-II, reduced expression of p62 and increased DQ-BSA dequenching suggest intact autophagy and functional lysosomes in A0H0 macrophages. Markedly decreased acid TG hydrolase activity and lipid flux independent of bafilomycin A1 treatment, however, argue against effective lysosomal degradation of LDs in A0H0 macrophages. We conclude that autophagy of proteins and cell organelles but not of LDs is active as a compensatory mechanism to circumvent and balance the reduced availability of energy substrates in A0H0 macrophages.


Assuntos
Autofagia/fisiologia , Lipólise/fisiologia , Macrófagos Peritoneais/metabolismo , Triglicerídeos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Catepsina B/biossíntese , Catepsina B/genética , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Lipase/genética , Lipase/metabolismo , Lipólise/efeitos dos fármacos , Lisossomos/enzimologia , Lisossomos/genética , Macrolídeos/farmacologia , Macrófagos Peritoneais/citologia , Camundongos , Camundongos Mutantes , Esterol Esterase/genética , Esterol Esterase/metabolismo , Triglicerídeos/genética
7.
Hum Mol Genet ; 24(15): 4198-211, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-25926625

RESUMO

Saposin (Sap) C deficiency is a rare variant form of Gaucher disease caused by impaired Sap C expression or accelerated degradation, and associated with accumulation of glucosylceramide and other lipids in the endo/lysosomal compartment. No effective therapies are currently available for the treatment of Sap C deficiency. We previously reported that a reduced amount and enzymatic activity of cathepsin (Cath) B and Cath D, and defective autophagy occur in Sap C-deficient fibroblasts. Here, we explored the use of two compounds, BCM-95, a curcumin derivative, and (2-hydroxypropyl)-ß-cyclodextrin (HP-ß-CD), to improve lysosomal function of Sap C-deficient fibroblasts. Immunofluorescence and biochemical studies documented that each compound promotes an increase of the expression levels and activities of Cath B and Cath D, and efficient clearance of cholesterol (Chol) and ceramide (Cer) in lysosomes. We provide evidence that BCM-95 and HP-ß-CD enhance lysosomal function promoting autophagic clearance capacity and lysosome reformation. Our findings suggest a novel pharmacological approach to Sap C deficiency directed to treat major secondary pathological aspects in this disorder.


Assuntos
Curcumina/efeitos adversos , Doença de Gaucher/tratamento farmacológico , Saposinas/genética , beta-Ciclodextrinas/administração & dosagem , Autofagia/efeitos dos fármacos , Catepsina B/biossíntese , Catepsina B/genética , Catepsina D/biossíntese , Catepsina D/genética , Curcumina/análogos & derivados , Fibroblastos/metabolismo , Fibroblastos/patologia , Doença de Gaucher/genética , Doença de Gaucher/patologia , Glucosilceramidas/metabolismo , Humanos , Lisossomos/genética , Lisossomos/patologia , Saposinas/deficiência
8.
Braz. dent. j ; 25(6): 502-507, Nov-Dec/2014. tab
Artigo em Inglês | LILACS | ID: lil-732258

RESUMO

This aim of this study was to assess the ability of manual or rotary instrumentation associated with photodynamic therapy (PDT) to reduce Enterococcus faecalis using three combinations of light/photosensitizers: toluidine blue O/laser, fuchsin/halogen light and fuchsin/LED. Twenty deciduous molars were selected and contaminated with Enterococcus faecalis (McFarland 0.5 scale). Working length determination was performed by visual method. The teeth were randomly divided into two groups: G1 (n=10): manual instrumentation (Kerr-type files) and G2 (n=10): rotary instrumentation (ProTaper system). The bacteria were collected three times using sterile paper cones compatible with the anatomic diameter of the root canal for 30 s before and after instrumentation and after PDT. The samples were diluted in peptone water, seeded on blood agar plates and incubated in an oven at 37 °C for colony-forming units counting. The decrease of E. faecalis counts after instrumentation and after PDT was compared using the Wilcoxon test, t-test and Kruskal Wallis test. A significant reduction of E. faecalis occurred after manual and rotary instrumentation and after PDT using the three combinations of light/photosensitizer (p<0.05). It may be concluded that both rotary and manual instrumentation reduced E. faecalis. Fuchsin with halogen light or LED irradiation and toluidine blue O with laser irradiation can be used to reduce E. faecalis in root canals of primary molars. PDT can be used as an adjuvant to conventional endodontic treatment.


O objetivo do presente estudo foi avaliar a redução de Enterococcus faecalis após instrumentação manual ou rotatória associada à terapia fotodinâmica (PDT) utilizando 3 combinações luz/fotossensibilizante: azul de toluidina O/laser, fucsina/luz halógena e fucsina/LED. Foram selecionados 20 molares decíduos que foram contaminados com Enterococcus faecalis (escala 0,5 de McFarland). A odontometria foi feita através do método visual. Os dentes foram divididos aleatoriamente em dois grupos: G1 (n=10): instrumentação manual (limas tipo Kerr) e G2 (n=10): instrumentação rotatória (sistema ProTaper). Foram realizadas coletas com cone de papel estéril compatível com o diâmetro anatômico do canal durante 30 s antes e após a instrumentação e a PDT. As amostras foram diluídas em água peptonada, semeadas em placas de agar-sangue e incubadas em estufa a 37 °C para contagem das unidades formadoras de colônias. As comparações antes da redução de E. faecalis após a instrumentação e após a realização da PDT foram realizadas pelo teste de Wilcoxon, teste t e Kruskal Wallis. Houve redução significante de E. faecalis após a instrumentação manual ou rotatória e após realização da PDT com as três combinações de luz/fotossensibilizante (p<0,05). Pode-se concluir que a instrumentação rotatória e manual acarretou a redução de E. faecalis. A fucsina irradiada com luz halógena ou led e o azul de toluidina irradiado com laser podem ser utilizados para redução de E. faecalis do sistema de canais radiculares de molares decíduos. A terapia fotodinâmica pode ser utilizada como coadjuvante ao tratamento endodôntico convencional.


Assuntos
Animais , Camundongos , Fosfatase Ácida/biossíntese , Catepsina B/biossíntese , Leucina/análogos & derivados , Leupeptinas/farmacologia , Melanoma Experimental/enzimologia , Oligopeptídeos/farmacologia , Pepstatinas/farmacologia , Peptídeo Hidrolases/biossíntese , Inibidores de Proteases/farmacologia , Indução Enzimática , Leucina/farmacologia , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologia , Células Tumorais Cultivadas
9.
J Biol Chem ; 289(31): 21716-26, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24939850

RESUMO

The induction of inflammatory cytokines such as IL-1ß is associated with the progression of human immunodeficiency virus, type 1 (HIV-1) disease or AIDS. Unlike most inflammatory cytokines that are regulated by NF-κB at the transcriptional level, production of mature IL-1ß also depends on inflammasome activation. The mechanism by which HIV-1 induces pro-IL-1ß expression and activates inflammasomes to cleave pro-IL-1ß into its bioactive form is not clearly defined. We report here that HIV-1 infection in human monocytes efficiently induced IL-1ß expression and inflammasome activation. Toll-like receptor 8 (TLR8) was required for inducing pro-IL-1ß expression, whereas the NLRP3 inflammasome was required for IL-1ß maturation and release. Furthermore, the lysosomal protease cathepsin B and HIV-1 induced production of reactive oxygen species were critical for HIV-induced inflammasome activation and IL-1ß production. HIV-1 entry, reverse transcription, and integration were all required for both pro-IL-1ß expression and inflammasome activation. Finally, we show that HIV-1-derived RNA was sufficient to induce both pro-IL-1ß expression and inflammasome activation. We conclude that HIV-1 infection induced the expression of pro-IL-1ß via TLR8-mediated mechanisms and activated caspase-1 through the NLRP3 inflammasome to cleave pro-IL-1ß into bioactive IL-1ß. These findings help to elucidate mechanisms of HIV-1 disease progression and identify novel targets for treating HIV-1 induced inflammation and immune activation.


Assuntos
Proteínas de Transporte/metabolismo , Infecções por HIV/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/biossíntese , Monócitos/metabolismo , Receptor 8 Toll-Like/fisiologia , Catepsina B/biossíntese , Técnicas de Silenciamento de Genes , HIV-1/genética , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR , RNA Viral/genética , Espécies Reativas de Oxigênio/metabolismo , Receptor 8 Toll-Like/genética
10.
Int J Mol Sci ; 15(4): 5807-20, 2014 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-24714089

RESUMO

Cathepsin B is one of the major lysosomal cysteine proteases involved in neuronal protein catabolism. This cathepsin is released after traumatic injury and increases neuronal death; however, release of cystatin C, a cathepsin inhibitor, appears to be a self-protective brain response. Here we describe the effect of cystatin C intracerebroventricular administration in rats prior to inducing a traumatic brain injury. We observed that cystatin C injection caused a dual response in post-traumatic brain injury recovery: higher doses (350 fmoles) increased bleeding and mortality, whereas lower doses (3.5 to 35 fmoles) decreased bleeding, neuronal damage and mortality. We also analyzed the expression of cathepsin B and cystatin C in the brains of control rats and of rats after a traumatic brain injury. Cathepsin B was detected in the brain stem, cerebellum, hippocampus and cerebral cortex of control rats. Cystatin C was localized to the choroid plexus, brain stem and cerebellum of control rats. Twenty-four hours after traumatic brain injury, we observed changes in both the expression and localization of both proteins in the cerebral cortex, hippocampus and brain stem. An early increase and intralysosomal expression of cystatin C after brain injury was associated with reduced neuronal damage.


Assuntos
Lesões Encefálicas/mortalidade , Lesões Encefálicas/patologia , Catepsina B/biossíntese , Cistatina C/farmacologia , Animais , Apoptose , Tronco Encefálico/metabolismo , Catepsina B/metabolismo , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Plexo Corióideo/metabolismo , Cistatina C/biossíntese , Hemorragia/induzido quimicamente , Hipocampo/metabolismo , Masculino , Neurônios/patologia , Ratos , Ratos Wistar
11.
Oncol Rep ; 31(2): 940-6, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24337203

RESUMO

Degradation of the extracellular matrix (ECM) is a critical step of tumor cell invasion and requires protease-dependent proteolysis focalized at the invadopodia where the proteolysis of the ECM occurs. Most of the extracellular proteases belong to serine- or metallo-proteases and the invadopodia is where protease activity is regulated. While recent data looking at global protease activity in the growth medium reported that their activity and role in invasion is dependent on Na+/H+ exchanger 1 (NHE1)-driven extracellular acidification, there is no data on this aspect at the invadopodia, and an open question remains whether this acid extracellular pH (pHe) activation of proteases in tumor cells occurs preferentially at invadopodia. We previously reported that the NHE1 is expressed in breast cancer invadopodia and that the NHE1­dependent acidification of the peri-invadopodial space is critical for ECM proteolysis. In the present study, using, for the first time, in situ zymography analysis, we demonstrated a concordance between NHE1 activity, extracellular acidification and protease activity at invadopodia to finely regulate ECM digestion. We demonstrated that: (i) ECM proteolysis taking place at invadopodia is driven by acidification of the peri-invadopodia microenvironment; (ii) that the proteases have a functional pHe optimum that is acidic; (iii) more than one protease is functioning to digest the ECM at these invadopodial sites of ECM proteolysis; and (iv) lowering pHe or inhibiting the NHE1 increases protease secretion while blocking protease activity changes NHE1 expression at the invadopodia.


Assuntos
Neoplasias da Mama/patologia , Proteínas de Transporte de Cátions/metabolismo , Extensões da Superfície Celular/metabolismo , Matriz Extracelular/metabolismo , Peptídeo Hidrolases/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Antiarrítmicos/farmacologia , Catepsina B/antagonistas & inibidores , Catepsina B/biossíntese , Catepsina B/metabolismo , Proteínas de Transporte de Cátions/antagonistas & inibidores , Linhagem Celular Tumoral , Matriz Extracelular/patologia , Feminino , Guanidinas/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Metaloproteinase 14 da Matriz/biossíntese , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Invasividade Neoplásica/patologia , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Sulfonas/farmacologia , Tiofenos/farmacologia
12.
Free Radic Biol Med ; 65: 1398-1407, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24103565

RESUMO

Protein disulfide isomerase (PDI) and its homologs are oxidoreductases facilitating protein folding in the ER. Endo-PDI (also termed ERp46) is highly expressed in endothelial cells. It belongs to the PDI family but its physiological function is largely unknown. We studied the role of Endo-PDI in endothelial angiogenic responses. Stimulation of human umbilical vein endothelial cells (with TNFα (10ng/ml) increased ERK1/2 phosphorylation. This effect was largely attenuated by Endo-PDI siRNA, whereas JNK and p38 MAP kinase phosphorylation was Endo-PDI independent. Similarly, TNFα-stimulated NF-κB signaling determined by IκBα degradation as well as TNFα-induced ICAM expression was unaffected by Endo-PDI siRNA. The action of Endo-PDI was not mediated by extracellular thiol exchange or cell surface PDI as demonstrated by nonpermeative inhibitors and PDI-neutralizing antibody. Moreover, exogenously added PDI failed to restore ERK1/2 activation after Endo-PDI knockdown. This suggests that Endo-PDI acts intracellularly potentially by maintaining the Ras/Raf/MEK/ERK pathway. Indeed, knockdown of Endo-PDI attenuated Ras activation measured by G-LISA and Raf phosphorylation. ERK activation influences gene expression by the transcriptional factor AP-1, which controls MMP-9 and cathepsin B, two proteases required for angiogenesis. TNFα-stimulated MMP-9 and cathepsin B induction was reduced by silencing of Endo-PDI. Accordingly, inhibition of cathepsin B or Endo-PDI siRNA blocked the TNFα-stimulated angiogenic response in the spheroid outgrowth assays. Moreover ex vivo tube formation and in vivo Matrigel angiogenesis in response to TNFα were attenuated by Endo-PDI siRNA. In conclusion, our study establishes Endo-PDI as a novel, important mediator of AP-1-driven gene expression and endothelial angiogenic function.


Assuntos
Células Endoteliais da Veia Umbilical Humana/enzimologia , Neovascularização Fisiológica/fisiologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Fator de Transcrição AP-1/genética , Fator de Necrose Tumoral alfa/farmacologia , Indutores da Angiogênese/antagonistas & inibidores , Indutores da Angiogênese/farmacologia , Catepsina B/antagonistas & inibidores , Catepsina B/biossíntese , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Células Cultivadas , Retículo Endoplasmático , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas I-kappa B , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Metaloproteinase 9 da Matriz/biossíntese , NADPH Oxidases , Inibidor de NF-kappaB alfa , Fosforilação , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/genética , Dobramento de Proteína , Interferência de RNA , RNA Interferente Pequeno , Esferoides Celulares , Tiorredoxina Dissulfeto Redutase , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ras/genética
13.
Free Radic Biol Med ; 65: 1155-1163, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23999505

RESUMO

Advanced glycation end product (AGE)-modified proteins are formed by the nonenzymatic glycation of free amino groups of proteins and, along with lipofuscin (a highly oxidized aggregate of covalently cross-linked proteins, sugars, and lipids), have been found to accumulate during aging and in several age-related diseases. As the in vivo effects of diet-derived AGEs or lipofuscin remain elusive, we sought to study the impact of oral administration of glucose-, fructose-, or ribose-modified albumin or of artificial lipofuscin in a genetically tractable model organism. We report herein that continuous feeding of young Drosophila flies with culture medium enriched in AGEs or in lipofuscin resulted in reduced locomotor performance and in accelerated rates of AGE-modified proteins and carbonylated proteins accumulation in the somatic tissues and hemolymph of flies, as well as in a significant reduction of flies health span and life span. These phenotypic effects were accompanied by reduced proteasome peptidase activities in both the hemolymph and the somatic tissues of flies and higher levels of oxidative stress; furthermore, oral administration of AGEs or lipofuscin in flies triggered an upregulation of the lysosomal cathepsin B, L activities. Finally, RNAi-mediated cathepsin D knockdown reduced flies longevity and significantly augmented the deleterious effects of AGEs and lipofuscin, indicating that lysosomal cathepsins reduce the toxicity of diet-derived AGEs or lipofuscin. Our in vivo studies demonstrate that chronic ingestion of AGEs or lipofuscin disrupts proteostasis and accelerates the functional decline that occurs with normal aging.


Assuntos
Envelhecimento/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Produtos Finais de Glicação Avançada/farmacologia , Lipofuscina/farmacologia , Dobramento de Proteína/efeitos dos fármacos , Albuminas/química , Animais , Animais Geneticamente Modificados , Catepsina B/biossíntese , Catepsina B/metabolismo , Catepsina D/genética , Catepsina L/biossíntese , Catepsina L/metabolismo , Dieta , Frutose/química , Glucose/química , Produtos Finais de Glicação Avançada/administração & dosagem , Produtos Finais de Glicação Avançada/química , Glicosilação , Lipofuscina/administração & dosagem , Lipofuscina/química , Longevidade/efeitos dos fármacos , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Interferência de RNA , RNA Interferente Pequeno , Ribose/química , Regulação para Cima
14.
Mol Cell Biol ; 33(21): 4308-20, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24001769

RESUMO

Sorting-related receptor with A-type repeats (SORLA) is a sorting receptor for the amyloid precursor protein (APP) that prevents breakdown of APP into Aß peptides, a hallmark of Alzheimer's disease (AD). Several cytosolic adaptors have been shown to interact with the cytoplasmic domain of SORLA, thereby controlling intracellular routing of SORLA/APP complexes in cell lines. However, the relevance of adaptor-mediated sorting of SORLA for amyloidogenic processes in vivo remained unexplored. We focused on the interaction of SORLA with phosphofurin acidic cluster sorting protein 1 (PACS1), an adaptor that shuttles proteins between the trans-Golgi network (TGN) and endosomes. By studying PACS1 knockdown in neuronal cell lines and investigating transgenic mice expressing a PACS1-binding-defective mutant form of SORLA, we found that disruption of SORLA and PACS1 interaction results in the inability of SORLA/APP complexes to sort to the TGN in neurons and in increased APP processing in the brain. Loss of PACS1 also impairs the proper expression of the cation-independent mannose 6-phosphate receptor and its target cathepsin B, a protease that breaks down Aß. Thus, our data identified the importance of PACS1-dependent protein sorting for amyloidogenic-burden control via both SORLA-dependent and SORLA-independent mechanisms.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas Relacionadas a Receptor de LDL/fisiologia , Proteínas de Membrana Transportadoras/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/enzimologia , Catepsina B/biossíntese , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Proteínas Relacionadas a Receptor de LDL/química , Proteínas de Membrana Transportadoras/química , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Neurônios/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Receptor IGF Tipo 2/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
15.
Oncol Rep ; 30(4): 1681-6, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23877402

RESUMO

Ac­Phe­Lys­PABC­DOX (PDOX) is a smart doxorubicin (DOX) prodrug designed to decrease toxicities while maintaining the potent anticancer effects of DOX. The present study aimed to elucidate the molecular mechanisms of action of PDOX using MGC­803 gastric cancer cells as a model. The cells were treated with both PDOX and DOX, and cytotoxicities, cell cycle analysis, reactive oxygen species (ROS) generation, mitochondrial damage and ERK1/2 signaling pathway alterations were studied. Abundant cathepsin B expression was observed in the MGC­803 cells, and treatment with PDOX and DOX triggered dose­dependent cytotoxicity and resulted in a significant reduction in cell viability. IC50 of PDOX and DOX was 14.9 and 4.9 µM, respectively. Both PDOX and DOX significantly decreased p­ERK1/2, increased ROS generation, reduced mitochondrial membrane potential, caused mitochondrial swelling and arrested the cell cycle at the G2/S phase, and these effects were more pronounced for PDOX than for DOX. PDOX and DOX have different mechanisms of action, particularly the mitochondria­centered intrinsic apoptosis involving reactive oxidative stress and the ERK1/2 signaling pathway.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Doxorrubicina/análogos & derivados , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oligopeptídeos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Catepsina B/biossíntese , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Dilatação Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
16.
Oncol Rep ; 30(2): 723-30, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23708264

RESUMO

The molecular mechanism involved in the metastasis of endometrial cancer (EC) remains unclear. The lysosomal cysteine protease Cathepsin B has been implicated in the progression of various human tumors. In the present study, we assessed the expression of Cathepsin B and its functions in EC. Immunohistochemistry was used to examine Cathepsin B expression in 76 paraffin-embedded endometrial tumor tissues. Lentiviral packing short hairpin RNA (shRNA) was transfected into HEC-1A cells to build a stable Cathepsin B knockdown cell line. The cellular levels of Cathepsin B mRNA and protein were detected by real-time PCR and western immunoblotting. The functions of Cathepsin B in EC cells were measured by MTT, migration and invasion assays. In additon, tumorigenicity assays were established in nude mice to study tumor growth in vivo. The results of our study showed that Cathepsin B was overexpressed in EC tissues compared with normal endometrium and endometrial atypical hyperplasia. Depletion of Cathepsin B in vitro inhibited cell proliferation, migration and invasion. Tumor formation assays confirmed that suppression of Cathepsin B inhibited the proliferation potential of HEC-1A cells in vivo, demonstrated by lower proliferation rates. These results suggest that Cathepsin B may act as an oncogene in EC, with the potential to provide a new therapeutic target for treating endometrial malignancy.


Assuntos
Catepsina B/deficiência , Catepsina B/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Animais , Testes de Carcinogenicidade/métodos , Catepsina B/biossíntese , Catepsina B/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Hiperplasia Endometrial/genética , Hiperplasia Endometrial/metabolismo , Hiperplasia Endometrial/patologia , Neoplasias do Endométrio/metabolismo , Endométrio/metabolismo , Endométrio/patologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , RNA Mensageiro/genética
17.
Int J Cancer ; 133(8): 1982-93, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23564480

RESUMO

Invasive pituitary adenomas (PAs) are generally refractory to conventional therapy and salvage treatment with temozolomide (TMZ). In addition to antiprotozoan effects, pyrimethamine (PYR) has recently shown its strong antitumor activity as an antineoplastic agent or in combination with TMZ in metastatic melanoma cells. In this study, the effects of TMZ, PYR or TMZ/PYR combination on rat/mouse PA cell lines αT3-1, GH3, MMQ and ATt-20 as well as GH3 xenograft tumor model were evaluated. TMZ/PYR combination synergistically inhibited proliferation, invasion and induced apoptosis of these PA cell lines in vitro. Strikingly, combination treatment with TMZ and PYR produced synergistic antitumor activity and enhanced the survival rate of GH3 xenograft tumor models without increasing systemic side effects. In addition, TMZ/PYR induced cell cycle arrest, increased DNA damage, upregulated the expression of cathepsin B, BAX, cleaved PARP and phosphorylated histone H2AX as well as elevated caspase3/7, 8 and 9 activities. The decreased expression of Bcl-2, MMP-2 and MMP-9 alone with cytochrome c release from mitochondria into the cytosol was also observed in the TMZ/PYR combination group. The increase in cell apoptosis due to combination with PYR was rescued by leucovorin. These data suggest that PYR may enhance the efficacy of TMZ via triggering both cathepsin B-dependent and caspase-dependent apoptotic pathways. Therefore, combination of PYR and TMZ may provide a novel regimen for invasive PAs refractory to standard therapy and TMZ.


Assuntos
Apoptose/efeitos dos fármacos , Catepsina B/metabolismo , Dacarbazina/análogos & derivados , Neoplasias Hipofisárias/tratamento farmacológico , Pirimetamina/farmacologia , Animais , Antineoplásicos Alquilantes/farmacologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Caspase 9/metabolismo , Catepsina B/biossíntese , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromos c/metabolismo , Dano ao DNA , Dacarbazina/farmacologia , Sinergismo Farmacológico , Feminino , Antagonistas do Ácido Fólico/farmacologia , Histonas/metabolismo , Leucovorina/farmacologia , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Invasividade Neoplásica , Transplante de Neoplasias , Fosforilação/efeitos dos fármacos , Neoplasias Hipofisárias/metabolismo , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Ratos , Temozolomida , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/biossíntese
18.
Stem Cells ; 31(1): 146-55, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23034897

RESUMO

Tumor tropism of human bone marrow-derived mesenchymal stem cells (MSC) has been exploited for the delivery of therapeutic genes for anticancer therapy. However, the exact contribution of these cells in the tumor microenvironment remains unknown. In this study, we examined the biological effect of MSC on tumor cells. The results showed that MSC inhibited the growth of human glioma cell lines and patient-derived primary glioma cells in vitro. Coadministration of MSC and glioma cells resulted in significant reduction in tumor volume and vascular density, which was not observed when glioma was injected with immortalized normal human astrocytes. Using endothelial progenitor cells (EPC) from healthy donors and HUVEC endothelial cells, the extent of EPC recruitment and capacity to form endothelial tubes was significantly impaired in conditioned media derived from MSC/glioma coculture, suggesting that MSC suppressed tumor angiogenesis through the release of antiangiogenic factors. Further studies using antibody array showed reduced expression of platelet-derived growth factor (PDGF)-BB and interleukin (IL)-1ß in MSC/glioma coculture when compared with controls. In MSC/glioma coculture, PDGF-BB mRNA and the corresponding proteins (soluble and membrane bound forms) as well as the receptors were found to be significantly downregulated when compared with that of glioma cocultured with normal human astrocytes or glioma monoculture. Furthermore, IL-1ß, phosphorylated Akt, and cathepsin B proteins were also reduced in MSC/glioma. Taken together, these data indicated that the antitumor effect of MSC may be mediated through downregulation of PDGF/PDGFR axis, which is known to play a key role in glioma angiogenesis. STEM Cells2013;31:146-155.


Assuntos
Glioma/patologia , Células-Tronco Mesenquimais/fisiologia , Neovascularização Patológica , Proteínas Proto-Oncogênicas c-sis/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Animais , Astrócitos , Becaplermina , Células da Medula Óssea/fisiologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/terapia , Catepsina B/biossíntese , Linhagem Celular Tumoral , Técnicas de Cocultura , Regulação para Baixo , Glioma/terapia , Células Endoteliais da Veia Umbilical Humana , Humanos , Interleucina-1beta/biossíntese , Transplante de Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-sis/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/biossíntese , Microambiente Tumoral
19.
PLoS One ; 7(2): e32272, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22384200

RESUMO

Cathepsin B (CTSB) is a proteolytic enzyme potentially modulating angiogenic processes and extracellular matrix remodeling. While matrix metalloproteinases are shown to be implicated in tissue fibrosis and vasculopathy associated with systemic sclerosis (SSc), the role of cathepsins in this disease has not been well studied. The aim of this study is to evaluate the roles of CTSB in SSc. Serum pro-CTSB levels were determined by enzyme-linked immunosorbent assay in 55 SSc patients and 19 normal controls. Since the deficiency of transcription factor Fli1 in endothelial cells is potentially associated with the development of SSc vasculopathy, cutaneous CTSB expression was evaluated by immunostaining in Fli1(+/-) and wild type mice as well as in SSc and control subjects. The effects of Fli1 gene silencing and transforming growth factor-ß (TGF-ß) on CTSB expression were determined by real-time PCR in human dermal microvascular endothelial cells (HDMECs) and dermal fibroblasts, respectively. Serum pro-CTSB levels were significantly higher in limited cutaneous SSc (lcSSc) and late-stage diffuse cutaneous SSc (dcSSc) patients than in healthy controls. In dcSSc, patients with increased serum pro-CTSB levels showed a significantly higher frequency of digital ulcers than those with normal levels. CTSB expression in dermal blood vessels was increased in Fli1(+/-) mice compared with wild type mice and in SSc patients compared with healthy controls. Consistently, Fli1 gene silencing increased CTSB expression in HDMECs. In cultured dermal fibroblasts from early dcSSc, CTSB expression was decreased compared with normal fibroblasts and significantly reversed by TGF-ß1 antisense oligonucleotide. In conclusion, up-regulation of endothelial CTSB due to Fli1 deficiency may contribute to the development of SSc vasculopathy, especially digital ulcers, while reduced expression of CTSB in lesional dermal fibroblasts is likely to be associated with skin sclerosis in early dcSSc.


Assuntos
Catepsina B/fisiologia , Escleroderma Sistêmico/patologia , Pele/patologia , Animais , Estudos de Casos e Controles , Catepsina B/biossíntese , Células Endoteliais/citologia , Ensaio de Imunoadsorção Enzimática/métodos , Matriz Extracelular/metabolismo , Feminino , Inativação Gênica , Humanos , Masculino , Camundongos , Microcirculação , Escleroderma Sistêmico/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Doenças Vasculares/patologia
20.
J Dermatol Sci ; 65(1): 58-62, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21945151

RESUMO

BACKGROUND: Keratolytic winter erythema (KWE) or Oudtshoorn skin disease is a rare autosomal dominant monogenic disorder of epidermal keratinisation characterized clinically by cyclical peeling of the palms and soles. Due to a founder effect many KWE families have been identified in South Africa and the gene has been localized to 8p23.1-22, but the causal gene has yet to be identified. OBJECTIVE: To examine two compelling positional and functional candidate genes within the critical region on 8p: cathepsin B (CTSB), a lysosomal cysteine protease localized to pericellular spaces between keratinocytes, possibly playing a role in cell-cell adhesion; and farnesyl-diphosphate farnesyltransferase (FDFT1), a membrane-associated enzyme in cholesterol biosynthesis which, among its many functions, plays a role in barrier permeability and integrity. METHOD: Mutation screening of the coding regions, 5'UTRs and intron/exon boundaries of CTSB and FDFT1 in genomic DNA and cDNA of patients affected with KWE. Relative gene expression profiles of CTSB and FDFT1 in palmoplantar skin biopsies were assessed by real-time RT-PCR. RESULTS: No DNA variants that segregate exclusively with KWE were identified. There was no significant difference in the CTSB expression profiles but a trend towards increased expression of FDFT1 was observed in the skin of affected individuals (p=0.063). This observation prompted analysis of the FDFT1 promoter region; however, no genetic variants segregating with the KWE phenotype were observed and it is likely that the increased expression was triggered in response to skin inflammation and peeling. CONCLUSION: CTSB and FDFT1 are excluded as candidates for KWE.


Assuntos
Catepsina B/biossíntese , Eritema/genética , Farnesil-Difosfato Farnesiltransferase/biossíntese , Ceratose/genética , Dermatopatias Genéticas/genética , Regiões 5' não Traduzidas , Catepsina B/genética , Catepsina B/metabolismo , Adesão Celular , Cromossomos Humanos Par 8 , Análise Mutacional de DNA , Eritema/metabolismo , Farnesil-Difosfato Farnesiltransferase/genética , Efeito Fundador , Perfilação da Expressão Gênica , Genes Dominantes , Variação Genética , Humanos , Ceratose/metabolismo , Modelos Genéticos , Mutação , Dermatopatias Genéticas/metabolismo , África do Sul
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