RESUMO
Heat shock protein 90 (HSP90) is a molecular chaperone that is up-regulated in cancer and is required for the folding of numerous signaling proteins. Consequently, HSP90 represents an ideal target for the development of new anti-cancer agents. The human HSP90 isoform, glucose-regulated protein 94 (GRP94), resides in the endoplasmic reticulum and regulates secretory pathways, integrins, and Toll-like receptors, which contribute to regulating immunity and metastasis. However, the cellular function of GRP94 remains underinvestigated. We report that GRP94 knockdown cells are defective in intracellular transport and, consequently, negatively impact the trafficking of F-actin toward the cellular cortex, integrin α2 and integrin αL toward the cell membrane and filopodia, and secretory vesicles containing the HSP90α-AHA1-survivin complex toward the leading edge. As a result, GRP94 knockdown cells form a multipolar spindle instead of bipolar morphology and consequently manifest a defect in cell migration and adhesion.
Assuntos
Movimento Celular , Polaridade Celular , Proteínas de Choque Térmico HSP90/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP90/genética , Humanos , Cadeias alfa de Integrinas/genética , Cadeias alfa de Integrinas/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Transporte Proteico , Fuso Acromático/genética , Fuso Acromático/metabolismoRESUMO
The hepatotoxicity of acetaminophen (APAP) is generally attributed to the formation of a reactive quinoneimine metabolite (NAPQI) that depletes glutathione and covalently binds to hepatocellular proteins. To explore the importance of the N-acyl group in APAP metabolism and toxicity, we synthesized 12 acyl side chain homologues of acetaminophen (APAP) and its 3'-regioisomer (AMAP), including the respective N-(4-pentynoyl) analogues PYPAP and PYMAP. Rat hepatocytes converted APAP, AMAP, PYPAP, and PYMAP extensively to O-glucuronide and O-sulfate conjugates in varying proportions, whereas glutathione or cysteine conjugates were observed only for APAP and PYPAP. PYPAP and PYMAP also underwent N-deacylation followed by O-sulfation and/or N-acetylation to a modest extent. The overall rates of metabolism in hepatocytes varied approximately 2-fold in the order APAP < AMAP ≈ PYPAP < PYMAP. Rat liver microsomes supplemented with NADPH and GSH converted APAP and PYPAP to their respective glutathione conjugates (formed via a reactive quinoneimine intermediate). With PYPAP only, a hydroxylated GSH conjugate was also observed. Thus, differences in biotransformation among these analogues were modest and mostly quantitative in nature. Cytotoxicity was evaluated in cultured hepatocytes by monitoring cell death using time-lapse photomicrography coupled with Hoechst 33342 and CellTox Green dyes to facilitate counting live cells vs dead cells, respectively. Progress curves for cell death and the areas under those curves showed that toxicity was markedly dependent on compound, concentration, and time. AMAP was essentially equipotent with APAP. Homologating the acyl side chain from C-2 to C-5 led to progressive increases in toxicity up to 80-fold in the para series. In conclusion, whereas N- or ring-substitution on APAP decrease metabolism and toxicity, homologating the N-acyl side chain increases metabolism about 2-fold, preserves the chemical reactivity of quinoneimine metabolites, and increases toxicity by up to 80-fold.
Assuntos
Acetaminofen/toxicidade , Acetaminofen/metabolismo , Animais , Biotransformação , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Isomerismo , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
A pressing need exists for autoimmune disease therapies that act in an antigen-specific manner while avoiding global immunosuppression. Multivalent soluble antigen arrays (SAgAPLP:LABL), designed to induce tolerance to a specific multiple sclerosis autoantigen, consist of a flexible hyaluronic acid (HA) polymer backbone cografted with multiple copies of autoantigen peptide (PLP) and cell adhesion inhibitor peptide (LABL). Previous in vivo studies revealed copresentation of both signals on HA was necessary for therapeutic efficacy. To elucidate therapeutic cellular mechanisms, in vitro studies were performed in a model B cell system to evaluate binding and specificity. Compared to HA and HA arrays containing only grafted PLP or LABL, SAgAPLP:LABL displaying both PLP and LABL exhibited greatly enhanced B cell binding. Furthermore, the binding avidity of SAgAPLP:LABL was primarily driven by the PLP antigen, determined via flow cytometry competitive dissociation studies. Fluorescence microscopy showed SAgAPLP:LABL induced mature receptor clustering that was faster than other HA arrays with only one type of grafted peptide. SAgAPLP:LABL molecules also reduced and inhibited IgM-stimulated signaling as discerned by a calcium flux assay. The molecular mechanisms of enhanced antigen-specific binding, mature receptor clustering, and dampened signaling observed in B cells may contribute to SAgAPLP:LABL therapeutic efficacy.
Assuntos
Autoantígenos/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Autoantígenos/química , Linfócitos B/imunologia , Linhagem Celular , Humanos , Ácido Hialurônico/química , Esclerose Múltipla/imunologia , Análise Serial de Proteínas , Transdução de SinaisRESUMO
Microalgae, with their high lipid content, are a promising feedstock for renewable fuels. Traditionally, human and environmentally toxic solvents have been used to extract these lipids, diminishing the sustainability of this process. Herein, pulsed electric field technology was utilized as a process intensification strategy to enhance lipid extraction from Ankistrodesmus falcatus wet biomass using the green solvent, ethyl acetate. The extraction efficiency for ethyl acetate without PEF was lower (83-88%) than chloroform. In addition, the ethyl acetate exhibited a 2-h induction period, while the chloroform showed no time dependence. Utilizing PEF technology resulted in 90% of the cells being lysed and a significant enhancement in the rate of lipid recovery using ethyl acetate. The increase in lipid recovery was due to the presence of the electric field and not due to temperature effects. The PEF technology uses less energy than other PEF systems reported in the literature.
Assuntos
Clorófitas/química , Eletroporação/métodos , Química Verde/métodos , Lipídeos/isolamento & purificação , Microalgas/química , Acetatos/química , Biomassa , Permeabilidade da Membrana Celular/efeitos da radiação , Clorofórmio/química , Cinética , Reprodutibilidade dos TestesRESUMO
Heat shock protein 90 (Hsp90) represents a promising therapeutic target for the treatment of cancer and other diseases. Unfortunately, results from clinical trials have been disappointing as off-target effects and toxicities have been observed. These detriments may be a consequence of pan-Hsp90 inhibition, as all clinically evaluated Hsp90 inhibitors simultaneously disrupt all four human Hsp90 isoforms. Using a structure-based approach, we designed an inhibitor of Grp94, the ER-resident Hsp90. The effect manifested by compound 2 on several Grp94 and Hsp90α/ß (cytosolic isoforms) clients were investigated. Compound 2 prevented intracellular trafficking of the Toll receptor, inhibited the secretion of IGF-II, affected the conformation of Grp94, and suppressed Drosophila larval growth, all Grp94-dependent processes. In contrast, compound 2 had no effect on cell viability or cytosolic Hsp90α/ß client proteins at similar concentrations. The design, synthesis, and evaluation of 2 are described herein.
Assuntos
Desenho de Fármacos , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular , Drosophila/efeitos dos fármacos , Drosophila/crescimento & desenvolvimento , Células HEK293 , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Proteínas de Membrana/química , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores Toll-Like/metabolismoRESUMO
A major limitation in the identification of novel antichlamydial compounds is the paucity of effective methods for large-scale compound screening. The immunofluorescence assay is the preferred approach for accurate quantification of the intracellular growth of Chlamydia. In this study, an immunofluorescence image-based method (termed image-based automated chlamydial identification and enumeration [iBAChIE]) was customized for fully automated quantification of Chlamydia infection using the freely available open-source image analysis software program CellProfiler and the complementary data exploration software program CellProfiler Analyst. The method yielded enumeration of different species and strains of Chlamydia highly comparably to the conventional manual methods while drastically reducing the analysis time. The inhibitory capability of established antichlamydial activity was also evaluated. Overall, these data support that iBAChIE is a highly effective tool for automated quantification of Chlamydia infection and assessment of antichlamydial activities of molecules. Furthermore, iBAChIE is expected to be amenable to high-throughput screening studies for inhibitory compounds and fluorescently labeled molecules to study host-pathogen interactions.
Assuntos
Antibacterianos/farmacologia , Infecções por Chlamydia/tratamento farmacológico , Infecções por Chlamydia/microbiologia , Chlamydia/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Imunofluorescência , Interações Hospedeiro-Patógeno , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Testes de Sensibilidade MicrobianaRESUMO
We have transfected murine neural stem cells (NSCs) and rat umbilical cord matrix-derived stem cells (RUCMSCs) with a plasmid expressing gaussia luciferase (gLuc). These cells are engineered to secrete the luciferase. We have used gLuc containing supernatant from culturing the NSCs to perform in vitro photodynamic therapy of murine melanoma cells (B16F10), and RUCMSCs to perform in vivo PDT of lung melanomas in C57BL/6 mice. The treatment system was comprised of aminolevulic acid as a prodrug for the synthesis of the photosensitizer protoporphyrin IX, gaussia luciferase, and its' substrate coelenterazine. A significant reduction of the number of live melanoma cells in vitro and a borderline significant retardation of tumour growth in vivo was observed after coelenterazine-mediated PDT.
Assuntos
Células-Tronco/metabolismo , Ácido Aminolevulínico/química , Ácido Aminolevulínico/farmacologia , Ácido Aminolevulínico/uso terapêutico , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Sangue Fetal/citologia , Imidazóis/química , Imidazóis/farmacologia , Luciferases/genética , Luciferases/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Oxirredução , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/uso terapêutico , Fármacos Fotossensibilizantes/toxicidade , Plasmídeos/metabolismo , Protoporfirinas/biossíntese , Protoporfirinas/uso terapêutico , Protoporfirinas/toxicidade , Pirazinas/química , Pirazinas/farmacologia , Ratos , Transplante de Células-Tronco , Células-Tronco/citologia , TransfecçãoRESUMO
Adenovirus serotype 4 (Ad4) is a major cause of Ad-associated human diseases. Ad4 is also considered to be a potential delivery vector for gene therapy. In this study, multiple spectroscopic techniques together with transmission electron microscopy (TEM) were employed to probe viral stability and to improve pharmaceutical formulations of Ad4-based vaccines and DNA carriers. Perturbations of secondary, tertiary and quaternary structure of Ad4 proteins induced by elevated temperatures over a wide pH range (3-8) were analyzed using circular dichroism, UV absorption and intrinsic and extrinsic fluorescence spectroscopy as well as static and dynamic light scattering. The spectroscopic results obtained indicate a decrease in Ad4 stability as pH increases from 4 to 8, similar to the behavior reported previously for Ad2 and Ad5, although the Ad4 virion appears to possess slightly more tolerance to thermal stress. An empirical phase diagram (EPD) approach was used to summarize the data in the form of a colored map. In addition, the different physical states of Ad4 identified by the EPD were confirmed by TEM images. The results obtained in this study reveal both structural similarities among three commonly employed Ad subtypes (2, 4 and 5) as well as unique properties of Ad4.
Assuntos
Adenoviridae/química , Espectrometria de Fluorescência/métodos , Proteínas Virais/química , Adenoviridae/ultraestrutura , Dicroísmo Circular/métodos , Temperatura Alta , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Transmissão , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectrofotometria Ultravioleta/métodos , Triptofano/química , Vírion/químicaRESUMO
Acute myeloid leukemia (AML) relapse results from the survival of chemotherapy-resistant and quiescent leukemia stem cells (LSC). These LSCs reside in the bone marrow microenvironment, comprised of other cells and extracellular matrix (ECM), which facilitates LSC quiescence through expression of cell adhesion molecules. We used decellularized Wharton's jelly matrix (DWJM), the gelatinous material in the umbilical cord, as a scaffolding material to culture leukemia cells, because it contains many components of the bone marrow extracellular matrix, including collagen, fibronectin, lumican, and hyaluronic acid (HA). Leukemia cells cultured in DWJM demonstrated decreased proliferation without undergoing significant differentiation. After culture in DWJM, these cells also exhibited changes in morphology, acquiring a spindle-shaped appearance, and an increase in the ALDH+ cell population. When treated with a high-dose of doxorubicin, leukemia cells in DWJM demonstrated less apoptosis compared with cells in suspension. Serial colony forming unit (CFU) assays indicated that leukemia cells cultured in DWJM showed increased colony-forming ability after both primary and secondary plating. Leukemia cell culture in DWJM was associated with increased N-cadherin expression by flow cytometry. Our data suggest that DWJM could serve as an ECM-based model to study AML stem cell-like cell behavior and chemotherapy sensitivity.
Assuntos
Proteínas da Matriz Extracelular/química , Matriz Extracelular/química , Leucemia Mieloide Aguda/metabolismo , Modelos Biológicos , Células-Tronco Neoplásicas/metabolismo , Geleia de Wharton/química , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/metabolismo , Células HL-60 , Humanos , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Geleia de Wharton/metabolismo , Geleia de Wharton/patologiaRESUMO
The morphology and affinities of newly discovered disc-shaped, soft-bodied fossils from the early Cambrian (Series 2: Stage 4, Dyeran) Carrara Formation are discussed. These specimens show some similarity to the Ordovician Discophyllum Hall, 1847; traditionally this taxon had been treated as a fossil porpitid. However, recently it has instead been referred to as another clade, the eldonids, which includes the enigmatic Eldonia Walcott, 1911 that was originally described from the Cambrian Burgess Shale. The status of various Proterozoic and Phanerozoic taxa previously referred to porpitids and eldonids is also briefly considered. To help ascertain that the specimens were not dubio- or pseudofossils, elemental mapping using energy dispersive X-ray spectroscopy (EDS) was conducted. This, in conjunction with the morphology of the specimens, indicated that the fossils were not hematite, iron sulfide, pyrolusite, or other abiologic mineral precipitates. Instead, their status as biologic structures and thus actual fossils is supported. Enrichment in the element carbon, and also possibly to some extent the elements magnesium and iron, seems to be playing some role in the preservation process.
RESUMO
BACKGROUND: Use of decellularized tissues has become popular in tissue engineering applications as the natural extracellular matrix can provide necessary physical cues that help induce the restoration and development of functional tissues. In relation to cochlear tissue engineering, the question of whether decellularized cochlear tissue can act as a scaffold and support the incorporation of exogenous cells has not been addressed. Investigators have explored the composition of the cochlear extracellular matrix and developed multiple strategies for decellularizing a variety of different tissues; however, no one has investigated whether decellularized cochlear tissue can support implantation of exogenous cells. METHODS: As a proof-of-concept study, human Wharton's jelly cells were perfused into decellularized cochleae isolated from C57BL/6 mice to determine if human Wharton's jelly cells could implant into decellularized cochlear tissue. Decellularization was verified through scanning electron microscopy. Cocheae were stained with DAPI and immunostained with Myosin VIIa to identify cells. Perfused cochleae were imaged using confocal microscopy. RESULTS: Features of the organ of Corti were clearly identified in the native cochleae when imaged with scanning electron microscopy and confocal microscopy. Acellular structures were identified in decellularized cochleae; however, no cellular structures or lipid membranes were present within the decellularized cochleae when imaged via scanning electron microscopy. Confocal microscopy revealed positive identification and adherence of cells in decellularized cochleae after perfusion with human Wharton's jelly cells. Some cells positively expressed Myosin VIIa after perfusion. CONCLUSIONS: Human Wharton's jelly cells are capable of successfully implanting into decellularized cochlear extracellular matrix. The identification of Myosin VIIa expression in human Wharton's jelly cells after implantation into the decellularized cochlear extracellular matrix suggest that components of the cochlear extracellular matrix may be involved in differentiation.
Assuntos
Cóclea/citologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Adesão Celular , Diferenciação Celular , Cóclea/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Feminino , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Miosina VIIa , Miosinas/genética , Miosinas/metabolismo , Transplante HeterólogoRESUMO
The herpes simplex virus type 1 (HSV-1) immediate-early phosphoprotein infected cell protein 0 (ICP0) is a potent transcriptional activator of viral genes and is required for efficient viral replication and reactivation from latency. However, it is largely unknown what role specific cellular factors play in the transactivator function of ICP0. With the long-term goal of identifying these factors, we developed a cell-based assay in a 96-well format to measure this activity of ICP0. We designed a system using a set of HSV-1 GFP reporter viruses in which the expression of GFP is potently induced by ICP0 in cell culture. The initial feasibility of this system was confirmed over a 24-h period by fluorescence microscopy. We adapted this assay to a 96-well plate format, quantifying GFP expression with a fluorescence scanner. Our results indicate that the cell-based assay we developed is a valid and effective method for examining the transactivating activity of ICP0. This assay can be used to identify cellular factors that regulate the transactivating activity of ICP0.
Assuntos
Técnicas Citológicas/métodos , Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Ativação Transcricional , Ubiquitina-Proteína Ligases/metabolismo , Fluorometria , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Células HeLa , Herpesvirus Humano 1/genética , Humanos , Coloração e Rotulagem/métodosRESUMO
Not all cells behave uniformly after treatment in tissue engineering studies. In fact, some treated cells display no signs of treatment or show unique characteristics not consistent with other treated cells. What if the "unique" cells could be isolated from a treated population, and further studied? Photo-convertible reporter proteins, such as Dendra2, allow for the ability to selectively identify unique cells with a secondary label within a primary labeled treated population. In the current study, select cells were identified and labeled through photo-conversion of Dendra2-transfected human Wharton's Jelly cells (hWJCs) for the first time. Robust photo-conversion of green-to-red fluorescence was achieved consistently in arbitrarily selected cells, allowing for precise cell identification of select hWJCs. The current study demonstrates a method that offers investigators the opportunity to selectively label and identify unique cells within a treated population for further study or isolation from the treatment population. Photo-convertible reporter proteins, such as Dendra2, offer the ability over non-photo-convertible reporter proteins, such as green fluorescent protein, to analyze unique individual cells within a treated population, which allows investigators to gain more meaningful information on how a treatment affects all cells within a target population.
RESUMO
Human Hsp90 isoforms are molecular chaperones that are often up-regulated in malignances and represent a primary target for Hsp90 inhibitors undergoing clinical evaluation. Hsp90α is a stress-inducible isoform of Hsp90 that plays a significant role in apoptosis and metastasis. Though Hsp90α is secreted into the extracellular space under metastatic conditions, its role in cancer biology is poorly understood. We report that Hsp90α associates with the Aha1 co-chaperone and found this complex to localize in secretory vesicles and at the leading edge of migrating cells. Knockdown of Hsp90α resulted in a defect in cell migration. The functional role of Hsp90α/Aha1 was studied by treating the cells with various novobiocin-based Hsp90 C-terminal inhibitors. These inhibitors disrupted the Hsp90α/Aha1 complex, caused a cytoplasmic redistribution of Hsp90α and Aha1, and decreased cell migration. Structure-function studies determined that disruption of Hsp90α/Aha1 association and inhibition of cell migration correlated with the presence of a benzamide side chain, since an acetamide substituted analog was less effective. Our results show that disruption of Hsp90α/Aha1 interactions with novobiocin-based Hsp90 C-terminal inhibitors may limit the metastatic potential of tumors.
Assuntos
Movimento Celular/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Chaperonas Moleculares/metabolismo , Novobiocina/farmacologia , Linhagem Celular , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Chaperonas Moleculares/genética , Ligação Proteica , Dobramento de Proteína , Isoformas de ProteínasRESUMO
An IgG2 monoclonal antibody (mAb) solution was subjected to stirring, generating high concentrations of nanometer and subvisible particles, which were then successfully size-enriched into different size bins by low-speed centrifugation or a combination of gravitational sedimentation and fluorescence-activated cell sorting (FACS). The size-fractionated mAb particles were assessed for their ability to elicit the release of cytokines from a population of donor-derived human peripheral blood mononuclear cells (PBMC) at two phases of the immune response. Fractions enriched in nanometer-sized particles showed a lower response than those enriched in micron-sized particles in this assay. Particles of 5-10 µm in size displayed elevated cytokine release profiles compared with other size ranges. Stir-stressed mAb particles had amorphous morphology, contained protein with partially altered secondary structure, elevated surface hydrophobicity (compared with controls), and trace levels of elemental fluorine. FACS size-enriched the mAb particle samples, yet did not notably alter the overall morphology or composition of particles as measured by microflow imaging, transmission electron microscopy, and scanning electron microscopy-energy dispersive X-ray spectroscopy. The utility and limitations of FACS for size separation of mAb particles and potential of in vitro PBMC studies to rank-order the immunogenic potential of various types of mAb particles are discussed.
Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Citometria de Fluxo/métodos , Tamanho da Partícula , Humanos , Imunoglobulina G/análise , Imunoglobulina G/química , Leucócitos Mononucleares/citologia , Microesferas , Nanopartículas/análise , Nanopartículas/químicaRESUMO
The transcription factor atonal homolog 1 (ATOH1) has multiple homologues that are functionally conserved across species and is responsible for the generation of sensory hair cells. To evaluate potential functional differences between homologues, human and mouse ATOH1 (HATH1 and MATH-1, respectively) were nonvirally delivered to human Wharton's jelly cells (hWJCs) for the first time. Delivery of HATH1 to hWJCs demonstrated superior expression of inner ear hair cell markers and characteristics than delivery of MATH-1. Inhibition of HES1 and HES5 signaling further increased the atonal effect. Transfection of hWJCs with HATH1 DNA, HES1 siRNA, and HES5 siRNA displayed positive identification of key hair cell and support cell markers found in the cochlea, as well as a variety of cell shapes, sizes, and features not native to hair cells, suggesting the need for further examination of other cell types induced by HATH1 expression. In the first side-by-side evaluation of HATH1 and MATH-1 in human cells, substantial differences were observed, suggesting that the two atonal homologues may not be interchangeable in human cells, and artificial expression of HATH1 in hWJCs requires further study. In the future, this line of research may lead to engineered systems that would allow for evaluation of drug ototoxicity or potentially even direct therapeutic use.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Técnicas de Reprogramação Celular/métodos , Células Ciliadas Auditivas Internas/citologia , Células-Tronco Mesenquimais/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem da Célula , Feminino , Corantes Fluorescentes/metabolismo , Vetores Genéticos/genética , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Recém-Nascido , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Miosina VIIa , Miosinas/biossíntese , Miosinas/genética , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Transdução de Sinais , Especificidade da Espécie , Fatores de Transcrição HES-1 , TransfecçãoRESUMO
Differentiating stem cells using gene delivery is a key strategy in tissue engineering and regenerative medicine applications. Nonviral gene delivery bypasses several safety concerns associated with viral gene delivery; however, leading nonviral techniques, such as electroporation, subject cells to high stress and can result in poor cell viabilities. Inhibition of Rho-associated coiled-coil kinase (ROCK) has been shown to mitigate apoptotic mechanisms associated with detachment and freezing of induced pluripotent stem cells and embryonic stem cells; however, inhibiting ROCK in mesenchymal stromal cells (MSCs) for improving gene delivery applications has not been reported previously. In this study, we hypothesized that ROCK Inhibitor (RI) would improve cell viability and gene expression in primary human umbilical cord mesenchymal stromal cells (hUCMSCs) when transfected via Nucleofection™. As hypothesized, the pre-treatment and post-treatment of hUCMSCs transfected via nucleofection with Y-27632-RI significantly improved survival rates of hUCMSCs and gene expression as measured by green fluorescent protein intensity. This study provides the first comparative look at the effect of Y-27632-RI on hUCMSCs that underwent transfection via nucleofection and shows that using Y-27632-RI in concert with nucleofection could greatly enhance the utility of differentiating and reprogramming hUCMSCs for tissue engineering applications.
Assuntos
Amidas/farmacologia , Células-Tronco Mesenquimais/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Transfecção , Cordão Umbilical/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Sobrevivência Celular , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologiaRESUMO
The long-term objective of this project is to utilize the I-domain protein for the α-subunit of LFA-1 to target drugs to lymphocytes by binding to ICAM receptors on the cell surface. The short-term goal is to provide proof-of-concept that I-domain conjugated to small molecules can still bind to and uptake by ICAM-1 on the surface of lymphocytes (i.e., Raji cells). To accomplish this goal, the I-domain protein was labeled with FITC at several lysine residues to produce the FITC-I-domain and CD spectroscopy showed that the FITC-I-domain has a secondary structure similar to that of the parent I-domain. The FITC-I-domain was taken up by Raji cells via receptor-mediated endocytosis and its uptake can be blocked by anti-I-domain mAb but not by its isotype control. Antibodies to ICAM-1 enhance the binding of I-domain to ICAM-1, suggesting it binds to ICAM-1 at different sites than the antibodies. The results indicate that fluorophore modification does not alter the binding and uptake properties of the I-domain protein. Thus, I-domain could be useful as a carrier of drug to target ICAM-1-expressing lymphocytes.
RESUMO
Aggregated alpha-synuclein and the point mutations Ala30Pro and Ala53Thr of alpha-synuclein are associated with Parkinson's disease. The physiological roles of alpha-synuclein and methionine oxidation of the alpha-synuclein protein structure and function are not fully understood. Methionine sulfoxide reductase A (MsrA) reduces methionine sulfoxide residues and functions as an antioxidant. To monitor the effect of methionine oxidation to alpha-synuclein on basic cellular processes, alpha-synucleins were expressed in msrA null mutant and wild-type yeast cells. Protein degradation was inhibited in the alpha-synuclein-expressing msrA null mutant cells compared to alpha-synuclein-expressing wild-type cells. Increased inhibition of degradation and elevated accumulations of fibrillated proteins were observed in SynA30P-expressing msrA null mutant cells. Additionally, methionine oxidation inhibited alpha-synuclein phosphorylation in yeast cells and in vitro by casein kinase 2. Thus, a compromised MsrA function combined with alpha-synuclein overexpression may promote processes leading to synucleinopathies.