RESUMO
A series of novel antimitotic agents was designed using the replacement of heterocyclic cores in two tubulin-targeting lead molecules with the acylated 4-aminoisoxazole moiety. Target compounds were synthesized via heterocyclization of ß-aryl-substituted vinylketones by tert-butyl nitrite in the presence of water as a key step. 4-Methyl-N-[5-methyl-3-(3,4,5-trimethoxyphenyl)isoxazol-4-yl]benzamide (1aa) was found to stimulate partial depolymerization of microtubules of human lung carcinoma A549 cells at a high concentration of 100 µM and to totally inhibit cell growth (IC50 = 0.99 µM) and cell viability (IC50 = 0.271 µM) in the nanomolar to submicromolar concentration range. These data provide evidence of the multitarget profile of the cytotoxic action of compound 1aa. The SAR study demonstrated that the 3,4,5-trimethoxyphenyl residue is the key structural parameter determining the efficiency both towards tubulin and other molecular targets. The cytotoxicity of 3-methyl-N-[5-methyl-3-(3,4,5-trimethoxyphenyl)isoxazol-4-yl]benzamide (1ab) to the androgen-sensitive human prostate adenocarcinoma cancer cell line LNCaP (IC50 = 0.301 µM) was approximately one order of magnitude higher than that to the conditionally normal cells lines WI-26 VA4 (IC50 = 2.26 µM) and human umbilical vein endothelial cells (IC50 = 5.58 µM) and significantly higher than that to primary fibroblasts (IC50 > 75 µM).
Assuntos
Antimitóticos , Antineoplásicos , Neoplasias , Antimitóticos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose , Benzamidas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Células Endoteliais/metabolismo , Humanos , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacologiaRESUMO
Glucan linked to proteins is a natural mega-glycoconjugate (mGC) playing the central role as a structural component of a yeast cell wall (CW). Regulation of functioning of non-covalently bound glucanosyltransglycosylases (ncGTGs) that have to remodel mGC to provide CW extension is poorly understood. We demonstrate that the main ncGTGs Bgl2 and Scw4 have phosphorylated and glutathionylated residues and are represented in CW as different pools of molecules having various firmness of attachment. Identified pools contain Bgl2 molecules with unmodified peptides, but differ from each other in the presence and combination of modified ones, as well as in the presence or absence of other CW proteins. Correlation of Bgl2 distribution among pools and its N-glycosylation was not found. Glutathione affects Bgl2 conformation, probably resulting in the mode of its attachment and enzymatic activity. Bgl2 from the pool of unmodified and monophosphorylated molecules demonstrates the ability to fibrillate after isolation from CW. Revealing of Bgl2 microcompartments and their mosaic arrangement summarized with the results obtained give the evidence that the functioning of ncGTGs in CW can be controlled by reversible post-translational modifications and facilitated due to their compact localization. The hypothetical scheme of distribution of Bgl2 inside CW is represented.
Assuntos
Parede Celular/metabolismo , Glucosiltransferases/metabolismo , Sequência de Aminoácidos/genética , Antifúngicos/metabolismo , Genes Fúngicos/genética , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Glucanos/metabolismo , Glucosidases/metabolismo , Glucosiltransferases/fisiologia , Glicosilação , Conformação Molecular , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transferases/metabolismoRESUMO
The use of metasurfaces operating in the terahertz regime as biosensor devices has attracted increased interest in recent years due to their enhanced sensitivity and more accurate detection capability. Typical designs are based on the replica of relatively simple unit cells, usually called metaatoms. In a previous paper, we proposed a new paradigm for ultrasensitive thin-film sensors based on complex unit cells, called generically metageometries or labyrinth metasurfaces. Here, we extend this concept towards biosensing, evaluating the performance of the labyrinth as a fungi detector. The sensing capabilities are numerically evaluated and a comparison with previous works in this field is performed, showing that metageometries improve the performance compared to metaatoms both in sensitivity and figure of merit, by a factor of more than four. In particular, we find that it is able to detect five fungi elements scattered on the unit cell, equivalent to a concentration of only 0.004/µm2.
Assuntos
Técnicas Biossensoriais/instrumentação , Análise Numérica Assistida por Computador , Eletricidade , Fungos/isolamento & purificação , Polipropilenos/químicaRESUMO
Subwavelength hole array (HA) metasurfaces support the so-called extraordinary optical transmission (EOT) resonance that has already been exploited for sensing. In this work, we demonstrate the superior performance of a different resonant regime of HA metasurfaces called anomalous EOT, by doing a thorough numerical and experimental study of its ability in thin-film label-free sensing applications in the terahertz (THz) band. A comprehensive analysis using both the regular and anomalous EOT resonances is done by depositing thin layers of dielectric analyte slabs of different thicknesses on the structures in different scenarios. We carry out a detailed comparison and demonstrate that the best sensing performance is achieved when the structure operates in the anomalous EOT resonance and the analyte is deposited on the non-patterned side of the metasurface, improving by a factor between 2 and 3 the results of the EOT resonance in any of the considered scenarios. This can be explained by the comparatively narrower linewidth of the anomalous EOT resonance. The results presented expand the reach of subwavelength HAs for sensing applications by considering the anomalous EOT regime that is usually overlooked in the literature.
RESUMO
Dyskeratosis congenita (DC) is an inherited multisystem disorder, characterized by oral leukoplakia, nail dystrophy, and abnormal skin pigmentation, as well as high rates of bone marrow (BM) failure, solid tumors, and other medical problems such as osteopenia. DC and telomere biology disorders (collectively referred to as TBD here) are caused by germline mutations in telomere biology genes leading to very short telomeres and limited proliferative potential of hematopoietic stem cells. We found that skeletal stem cells (SSCs) within the BM stromal cell population (BMSCs, also known as BM-derived mesenchymal stem cells), may contribute to the hematologic phenotype. TBD-BMSCs exhibited reduced clonogenicity, spontaneous differentiation into adipocytes and fibrotic cells, and increased senescence in vitro. Upon in vivo transplantation into mice, TBD-BMSCs failed to form bone or support hematopoiesis, unlike normal BMSCs. TERC reduction (a TBD-associated gene) in normal BMSCs by small interfering TERC-RNA (siTERC-RNA) recapitulated the TBD-BMSC phenotype by reducing proliferation and secondary colony-forming efficiency, and by accelerating senescence in vitro. Microarray profiles of control and siTERC-BMSCs showed decreased hematopoietic factors at the messenger RNA level and decreased secretion of factors at the protein level. These findings are consistent with defects in SSCs/BMSCs contributing to BM failure in TBD.
Assuntos
Células da Medula Óssea/metabolismo , Disceratose Congênita/genética , Células-Tronco Mesenquimais/metabolismo , RNA/genética , Telomerase/genética , Telômero/metabolismo , Adolescente , Adulto , Animais , Sequência de Bases , Células da Medula Óssea/patologia , Diferenciação Celular , Proliferação de Células , Senescência Celular , Criança , Pré-Escolar , Ensaio de Unidades Formadoras de Colônias , DNA Helicases/genética , DNA Helicases/metabolismo , Disceratose Congênita/patologia , Feminino , Hematopoese/genética , Humanos , Masculino , Células-Tronco Mesenquimais/patologia , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , RNA/antagonistas & inibidores , RNA/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Telomerase/antagonistas & inibidores , Telomerase/metabolismo , Telômero/química , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Tubuloclustin [N-(7-adamant-2-yloxy-7-oxoheptanoyl)-N-deacetylcolchicine], a highly cytotoxic anti-tubulin compound is known for its ability to promote microtubule disassembly followed by the formation of tubulin clusters of unique morphology. Three series of antimitotic agents related to tubuloclustin were designed and synthesized in order to enhance the molecular diversity of "tubuloclustin-like" family of compounds. The series of compounds with modified adamantane moiety was highly potent in cytotoxic effect on human lung carcinoma A549 cells (EC50 = 6-400 nM) and was active in affecting the microtubule arrays and induction of strong tubulin clusterization. In two other sets of compounds, the colchicine moiety of tubuloclustin was replaced by podophyllotoxin or combretastatin A-4. All combretastatin A-4 derivatives displayed noticeable cytotoxic activity ([Formula: see text]) but their effect on microtubules depended on the position of the linker attachment. Podophyllotoxin derivatives were also toxic to A549 cells ([Formula: see text]) and caused both microtubule depolymerization and some tubulin clustering. The data obtained gave additional evidence that the whole panel of C7-colchicine, podophyllotoxin and combretastatin derivatives could manifest clustering effect, and the strength of this effect correlated with cytotoxic activity of the compounds.
Assuntos
Adamantano/análogos & derivados , Antimitóticos/síntese química , Colchicina/análogos & derivados , Tubulina (Proteína)/metabolismo , Células A549 , Adamantano/química , Antimitóticos/química , Antimitóticos/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colchicina/química , Humanos , Modelos Moleculares , Estrutura Molecular , Tubulina (Proteína)/químicaRESUMO
Emerging evidence suggests that reactive oxygen species (ROS) can stimulate the Wnt/ß-catenin pathway in a number of cellular processes. However, potential sources of endogenous ROS have not been thoroughly explored. Here, we show that growth factor depletion in human neural progenitor cells induces ROS production in mitochondria. Elevated ROS levels augment activation of Wnt/ß-catenin signaling that regulates neural differentiation. We find that growth factor depletion stimulates the release of Ca(2+) from the endoplasmic reticulum stores. Ca(2+) subsequently accumulates in the mitochondria and triggers ROS production. The inhibition of mitochondrial Ca(2+) uptake with simultaneous growth factor depletion prevents the rise in ROS metabolism. Moreover, low ROS levels block the dissociation of the Wnt effector Dishevelled from nucleoredoxin. Attenuation of the response amplitudes of pathway effectors delays the onset of the Wnt/ß-catenin pathway activation and results in markedly impaired neuronal differentiation. Our findings reveal Ca(2+)-mediated ROS metabolic cues that fine-tune the efficiency of cell differentiation by modulating the extent of the Wnt/ß-catenin signaling output.
Assuntos
Cálcio/metabolismo , Diferenciação Celular , Mitocôndrias/metabolismo , Células-Tronco Neurais/citologia , Espécies Reativas de Oxigênio/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Humanos , Células-Tronco Neurais/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMO
BACKGROUND AIMS: Ex vivo expansion and serial passage of human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) is required to obtain sufficient quantities for clinical therapy. The BMSC confluence criteria used to determine passage and harvest timing vary widely, and the impact of confluence on BMSC properties remains controversial. The effects of confluence on BMSC properties were studied and confluence-associated markers were identified. METHODS: BMSC characteristics were analyzed as they grew from 50% to 100% confluence, including viability, population doubling time, apoptosis, colony formation, immunosuppression, surface marker expression, global gene expression and microRNA expression. In addition, culture supernatant protein, glucose, lactate and pH levels were analyzed. RESULTS: Confluence-dependent changes were detected in the expression of several cell surface markers: 39 culture supernatant proteins, 26 microRNAs and 2078 genes. Many of these surface markers, proteins, microRNAs and genes have been reported to be important in BMSC function. The pigment epithelium-derived factor/vascular endothelial growth factor ratio increased with confluence, but 80% and 100% confluent BMSCs demonstrated a similar level of immunosuppression of mixed lymphocyte reactions. In addition, changes in lactate and glucose levels correlated with BMSC density. CONCLUSIONS: BMSC characteristics change as confluence increases. 100% confluent BMSCs may have compromised pro-angiogenesis properties but may retain their immunomodulatory properties. Supernatant lactate and glucose levels can be used to estimate confluence and ensure consistency in passage and harvest timing. Flow cytometry or microRNA expression can be used to confirm that the BMSCs have been harvested at the appropriate confluence.
Assuntos
Células da Medula Óssea/citologia , Técnicas de Cultura de Células , Proliferação de Células/fisiologia , Células-Tronco Mesenquimais/citologia , Apoptose/fisiologia , Biomarcadores/metabolismo , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Proteínas do Olho/metabolismo , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica , Glucose/metabolismo , Humanos , Ácido Láctico/metabolismo , Masculino , Proteínas de Membrana/metabolismo , MicroRNAs/biossíntese , MicroRNAs/genética , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The role of actin, class I myosins and dynamin in endocytic uptake processes is well characterized, but their role during endo-phagosomal membrane trafficking and maturation is less clear. In Dictyostelium, knockout of myosin IB (myoB) leads to a defect in membrane protein recycling from endosomes back to the plasma membrane. Here, we show that actin plays a central role in the morphology and function of the endocytic pathway. Indeed, latrunculin B (LatB) induces endosome tubulation, a phenotype also observed in dynamin A (dymA)-null cells. Knockout of dymA impairs phagosome acidification, whereas knockout of myoB delays reneutralization, a phenotype mimicked by a low dose of LatB. As a read out for actin-dependent processes during maturation, we monitored the capacity of purified phagosomes to bind F-actin in vitro, and correlated this with the presence of actin-binding and membrane-trafficking proteins. Phagosomes isolated from myoB-null cells showed an increased binding to F-actin, especially late phagosomes. In contrast, early phagosomes from dymA-null cells showed reduced binding to F-actin while late phagosomes were unaffected. We provide evidence that Abp1 is the main F-actin-binding protein in this assay and is central for the interplay between DymA and MyoB during phagosome maturation.
Assuntos
Actinas/metabolismo , Dinaminas/metabolismo , Endossomos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Miosina Tipo I/metabolismo , Fagossomos/metabolismo , Proteínas de Protozoários/metabolismo , Western Blotting , Dictyostelium/metabolismo , Dictyostelium/ultraestrutura , Dinaminas/genética , Endossomos/ultraestrutura , Técnicas de Inativação de Genes , Modelos Biológicos , Miosina Tipo I/genética , Fagocitose , Fagossomos/ultraestrutura , Transporte Proteico , Proteínas de Protozoários/genéticaRESUMO
BACKGROUND: The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, brain, and other tissues. It has been hypothesized that the syndrome is caused by somatic mosaicism for a mutation that is lethal in the nonmosaic state. METHODS: We performed exome sequencing of DNA from biopsy samples obtained from patients with the Proteus syndrome and compared the resultant DNA sequences with those of unaffected tissues obtained from the same patients. We confirmed and extended an observed association, using a custom restriction-enzyme assay to analyze the DNA in 158 samples from 29 patients with the Proteus syndrome. We then assayed activation of the AKT protein in affected tissues, using phosphorylation-specific antibodies on Western blots. RESULTS: Of 29 patients with the Proteus syndrome, 26 had a somatic activating mutation (c.49GâA, p.Glu17Lys) in the oncogene AKT1, encoding the AKT1 kinase, an enzyme known to mediate processes such as cell proliferation and apoptosis. Tissues and cell lines from patients with the Proteus syndrome harbored admixtures of mutant alleles that ranged from 1% to approximately 50%. Mutant cell lines showed greater AKT phosphorylation than did control cell lines. A pair of single-cell clones that were established from the same starting culture and differed with respect to their mutation status had different levels of AKT phosphorylation. CONCLUSIONS: The Proteus syndrome is caused by a somatic activating mutation in AKT1, proving the hypothesis of somatic mosaicism and implicating activation of the PI3K-AKT pathway in the characteristic clinical findings of overgrowth and tumor susceptibility in this disorder. (Funded by the Intramural Research Program of the National Human Genome Research Institute.).
Assuntos
Mosaicismo , Mutação , Síndrome de Proteu/genética , Proteínas Proto-Oncogênicas c-akt/genética , Criança , Análise Mutacional de DNA , Éxons/genética , Genótipo , Humanos , Masculino , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Skeletal dysplasias are common disabling disorders characterized by aberrant growth of bone and cartilage leading to abnormal skeletal structures and functions, often attributable to defects in skeletal progenitor cells. The underlying molecular and cellular mechanisms of most skeletal dysplasias remain elusive. Although the Wnt/ß-catenin signaling pathway is required for skeletal progenitor cells to differentiate along the osteoblastic lineage, inappropriately elevated levels of signaling can also inhibit bone formation by suppressing osteoblast maturation. Here, we investigate interactions of the four major Gα protein families (Gα(s), Gα(i/o), Gα(q/11), and Gα(12/13)) with the Wnt/ß-catenin signaling pathway and identify a causative role of Wnt/ß-catenin signaling in fibrous dysplasia (FD) of bone, a disease that exhibits abnormal differentiation of skeletal progenitor cells. The activating Gα(s) mutations that cause FD potentiated Wnt/ß-catenin signaling, and removal of Gα(s) led to reduced Wnt/ß-catenin signaling and decreased bone formation. We further show that activation of Wnt/ß-catenin signaling in osteoblast progenitors results in an FD-like phenotype and reduction of ß-catenin levels rescued differentiation defects of FD patient-derived stromal cells. Gα proteins may act at the level of ß-catenin destruction complex assembly by binding Axin. Our results indicate that activated Gα proteins differentially regulate Wnt/ß-catenin signaling but, importantly, are not required core components of Wnt/ß-catenin signaling. Our data suggest that activated Gα proteins are playing physiologically significant roles during both skeletal development and disease by modulating Wnt/ß-catenin signaling strength.
Assuntos
Displasia Fibrosa Óssea/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Via de Sinalização Wnt , Adulto , Animais , Células da Medula Óssea/patologia , Displasia Fibrosa Óssea/patologia , Displasia Fibrosa Poliostótica/metabolismo , Displasia Fibrosa Poliostótica/patologia , Humanos , Camundongos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fenótipo , Células-Tronco/metabolismo , Células-Tronco/patologia , Células Estromais/metabolismo , Células Estromais/patologia , Regulação para Cima , beta Catenina/metabolismoRESUMO
The mechanisms by which bone marrow stromal cells (BMSCs) maintain multilineage potency in vitro remain elusive. To identify the transcriptional regulatory circuits that contribute to BMSC multipotency, we performed paired single-nucleus multiomics of the expansion of freshly isolated BMSCs and of BMSCs undergoing tri-lineage differentiation. By computationally reconstructing the regulatory programs associated with initial stages of differentiation and early expansion, we identified the TEAD family of transcription factors, which is inhibited by Hippo signaling, as highly active in the BMSC in vitro multipotent state. Pharmacological inhibition of TEAD enhanced BMSC osteogenic and adipogenic differentiation, whereas its activation maintained BMSCs in an undifferentiated state, supporting a model whereby isolation of BMSCs coincides with a TEAD-controlled transcriptional state linked to multipotency. Our study highlights the Hippo pathway as a pivotal regulator of BMSC multipotency, and our regulatory network inferences are a reservoir of testable hypotheses that link transcription factors and their regulons to specific aspects of BMSC behavior.
RESUMO
It was suggested that gap junctional intercellular communication (GJIC) and connexin (Cx) proteins play a crucial role in cell proliferation and differentiation. However, the mechanisms of cell coupling in regulating cell fate during embryonic development are poorly understood. To study the role of GJIC in proliferation and differentiation, we used a human neural progenitor cell line derived from the ventral mesencephalon. Fluorescence recovery after photobleaching (FRAP) showed that dye coupling was extensive in proliferating cells but diminished after the induction of differentiation, as indicated by a 2.5-fold increase of the half-time of fluorescence recovery. Notably, recovery half-time decreased strongly (five-fold) in the later stage of differentiation. Western blot analysis revealed a similar time-dependent expression profile of Cx43, acting as the main gap junction-forming protein. Interestingly, large amounts of cytoplasmic Cx43 were retained mainly in the Golgi network during proliferation but decreased when differentiation was induced. Furthermore, down-regulation of Cx43 by small interfering RNA reduced functional cell coupling, which in turn resulted in a 50% decrease of both the proliferation rate and neuronal differentiation. Our findings suggest a dual function of Cx43 and GJIC in the neural development of ReNcell VM197 human progenitor cells. GJIC accompanied by high Cx43 expression is necessary (1) to maintain cells in a proliferative state and (2) to complete neuronal differentiation, including the establishment of a neural network. However, uncoupling of cells is crucial in the early stage of differentiation during cell fate commitment.
Assuntos
Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Junções Intercelulares/fisiologia , Células-Tronco Neurais/citologia , Neurônios/citologia , Comunicação Celular , Linhagem Celular , HumanosRESUMO
Remodelling of the actin cytoskeleton plays a key role in particle internalisation and the phagosome maturation processes. Actin-binding proteins (ABPs) are the main players in actin remodelling but the precise role of these proteins in phagocytosis needs to be clarified. Annexins, a group of ABPs, are known to be present on phagosomes. Here, we identified annexin A1 as a factor that binds to isolated latex bead phagosomes (LBPs) in the presence of Ca(2+) and facilitates the F-actin-LBP interaction in vitro. In macrophages the association of endogenous annexin A1 with LBP membranes was strongly correlated with the spatial and temporal accumulation of F-actin at the LBP. Annexin A1 was found on phagocytic cups and around early phagosomes, where the F-actin was prominently concentrated. After uptake was completed, annexin A1, along with F-actin, dissociated from the nascent LBP surface. At later stages of phagocytosis annexin A1 transiently concentrated only around those LBPs that showed transient F-actin accumulation ('actin flashing'). Downregulation of annexin A1 expression resulted in impaired phagocytosis and actin flashing. These data identify annexin A1 as an important component of phagocytosis that appears to link actin accumulation to different steps of phagosome formation.
Assuntos
Citoesqueleto de Actina/metabolismo , Anexina A1/metabolismo , Fagocitose , Fagossomos/metabolismo , Citoesqueleto de Actina/genética , Actinas/metabolismo , Animais , Anexina A1/genética , Linhagem Celular , Camundongos , Ligação ProteicaRESUMO
Highly cytotoxic C7-modified colchicine analogues, exemplified by tubuloclustin, promote microtubule disassembly followed by the formation of very stable tubulin clusters, both in vitro and in cells. The proposed mechanism of action of tubuloclustin and its analogues, beyond that of colchicine, includes additional specific interactions with the α-tubulin subunit.
Assuntos
Adamantano/análogos & derivados , Colchicina/análogos & derivados , Colchicina/farmacologia , Tubulina (Proteína)/metabolismo , Adamantano/química , Adamantano/farmacologia , Animais , Células Cultivadas , Colchicina/química , Citotoxinas/química , Citotoxinas/farmacologia , Citometria de Fluxo , Imunofluorescência , Células HeLa , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Estrutura Molecular , Ligação Proteica/efeitos dos fármacosRESUMO
Hormone- and neuropeptide-containing secretory granules (SGs) of neuroendocrine PC12 cells are formed at the trans- Golgi network as immature SGs. These intermediates are converted to mature SGs in a complex maturation process, including matrix condensation, processing of cargo proteins and removal of proteins and membrane in clathrin-coated vesicles. The resulting mature SGs undergo Ca2+-dependent exocytosis upon an appropriate stimulus. We here show that the motor protein myosin Va is implicated in a maturation step of SGs, their binding to F-actin and their stimulated exocytosis. Interference with myosin Va function blocked the removal of the transmembrane protein furin from maturing SGs without affecting condensation and processing of proteins of the SG lumen. Furthermore, the ATP-inhibited binding of SGs to F-actin decreased with progressive maturation and upon interference with myosin Va function. Moreover, the expression of a dominant-negative myosin Va-tail or shRNA-based downregulation of myosin Va interfered with stimulated exocytosis of SGs. In summary,our data suggest an essential function of myosin Va in the membrane remodeling of SGs during maturation and a role in their exocytosis.
Assuntos
Exocitose/fisiologia , Vesículas Secretórias/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Cálcio/metabolismo , Estruturas Celulares/metabolismo , Vesículas Revestidas por Clatrina , Furina/metabolismo , Proteínas de Membrana/metabolismo , Membranas/metabolismo , Células PC12 , Ratos , Vesículas Secretórias/metabolismoRESUMO
BACKGROUND: Bone marrow stromal cells (BMSCs) are being used to treat a variety of conditions. For many applications a supply of cryopreserved products that can be used for acute therapy is needed. The establishment of a bank of BMSC products from healthy third party donors is described. METHODS: The recruitment of healthy subjects willing to donate marrow for BMSC production and the Good Manufacturing Practices (GMP) used for assessing potential donors, collecting marrow, culturing BMSCs and BMSC cryopreservation are described. RESULTS: Seventeen subjects were enrolled in our marrow collection protocol for BMSC production. Six of the 17 subjects were found to be ineligible during the donor screening process and one became ill and their donation was cancelled. Approximately 12 ml of marrow was aspirated from one posterior iliac crest of 10 donors; one donor donated twice. The BMSCs were initially cultured in T-75 flasks and then expanded for three passages in multilayer cell factories. The final BMSC product was packaged into units of 100 × 106 viable cells, cryopreserved and stored in a vapor phase liquid nitrogen tank under continuous monitoring. BMSC products meeting all lot release criteria were obtained from 8 of the 11 marrow collections. The rate of growth of the primary cultures was similar for all products except those generated from the two oldest donors. One lot did not meet the criteria for final release; its CD34 antigen expression was greater than the cut off set at 5%. The mean number of BMSC units obtained from each donor was 17 and ranged from 3 to 40. CONCLUSIONS: The production of large numbers of BMSCs from bone marrow aspirates of healthy donors is feasible, but is limited by the high number of donors that did not meet eligibility criteria and products that did not meet lot release criteria.
Assuntos
Células da Medula Óssea/citologia , Bancos de Tecidos , Preservação de Tecido/métodos , Adulto , Idoso , Envelhecimento/fisiologia , Antígenos CD34/metabolismo , Contagem de Células , Núcleo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Feminino , Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Células Estromais/citologia , Fatores de Tempo , Doadores de Tecidos , Adulto JovemRESUMO
BACKGROUND AIMS: Bone marrow stromal cells (BMSC) are being used for immune modulatory, anti-inflammatory and tissue engineering applications, but the properties responsible for these effects are not completely understood. Human BMSC were characterized to identify factors that might be responsible for their clinical effects and biomarkers for assessing their quality. METHODS: Early passage BMSC prepared from marrow aspirates of seven healthy subjects were compared with three human embryonic stem cell (hESC) samples, CD34(+) cells from three healthy subjects and three fibroblast cell lines. The cells were analyzed with oligonucleotide expression microarrays with more than 35 000 probes. RESULTS: BMSC gene expression signatures of BMSC differed from those of hematopoietic stem cells (HSC), hESC and fibroblasts. Genes upregulated in BMSC were involved with cell movement, cell-to-cell signaling and interaction and proliferation. The upregulated genes most probably belonged to pathways for integrin signaling, integrin-linked kinase (ILK) signaling, NF-E2-related factor-2 (NFR2)-mediated oxidative stress response, regulation of actin-based motility by Rho, actin cytoskeletal signaling, caveolar-mediated endocytosis, clathrin-mediated endocytosis and Wingless-type MMTV integration site (Wnt/ß catenin signaling. Among the most highly upregulated genes were structural extracellular matrix (ECM) proteins (α5 and ß5 integrin chains, fibronectin and collagen type IIIα1 and Vα1) and functional EMC proteins [connective tissue growth factor (CTGF), transforming growth factor beta-induced protein (TGFBI) and A disintegrin and metalloproteinase (ADAM12)]. CONCLUSIONS: Global analysis of human BMSC suggests that they are mobile, metabolically active, proliferative and interactive cells that make use of integrins and integrin signaling. They produce abundant ECM proteins that may contribute to their clinical immune modulatory and anti-inflammatory effects.
Assuntos
Células da Medula Óssea/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Células Estromais/metabolismo , Proteínas ADAM/genética , Proteína ADAM12 , Adulto , Células da Medula Óssea/citologia , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/genética , Proteínas da Matriz Extracelular/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Proteínas de Membrana/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Fator 2 Relacionado a NF-E2/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Células Estromais/citologia , Fator de Crescimento Transformador beta/genéticaRESUMO
A series of analogues of conjugate 1, combining an adamantane-based paclitaxel (taxol) mimetic with colchicine was synthesized and tested for cytotoxicity in a cell-based assay with the human lung carcinoma cell line A549. The most active compounds (10 EC(50) 2 ± 1.0 nM, 23 EC(50) 6 ± 1.4 nM, 26 EC(50) 5 ± 1.8 nM, 28 EC(50) 11 ± 1.7 nM, 30 EC(50) 4.8 ± 0.5 nM) were found to interfere with the microtubule dynamics in an interesting manner. Treatment of the cells with these compounds promoted disassembly of microtubules followed by the formation of stable tubulin clusters. Structure-activity relationships for the analogues of 23 revealed the sensitivity of both cytotoxicity and tubulin clustering ability to the linker length. The presence of adamantane (or another bulky hydrophobic and non-aromatic moiety) in 23 was found to play an important role in the formation of tubulin clusters. Structural requirements for optimal activity have been partially explained by molecular modeling.
Assuntos
Adamantano/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Colchicina/farmacologia , Microtúbulos/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Adamantano/química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Colchicina/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Microtúbulos/metabolismo , Modelos Moleculares , Estrutura Molecular , Paclitaxel/química , Paclitaxel/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Determining the distribution of specific binding sites on biological samples with high spatial accuracy (in the order of several nanometer) is an important challenge in many fields of biological science. Combination of high-resolution atomic force microscope (AFM) topography imaging with single-molecule force spectroscopy provides a unique possibility for the detection of specific molecular recognition events. The identification and localization of specific receptor binding sites on complex heterogeneous biosurfaces such as cells and membranes are of particular interest in this context. Simultaneous topography and recognition imaging was used to unravel the nanolandscape of cells of the immune system such as macrophages. The most studied phagocytic receptors include the Fc receptors that bind to the Fc portion of immunoglobulins. Here, nanomapping of FcγRs (Fc receptors for immunoglobulin G (IgG)) was performed on fixed J774.A1 mouse macrophage cell surfaces with magnetically coated AFM tips functionalized with Fc fragments of mouse IgG via long and flexible poly(ethylene glycol) linkers. Because of possible AFM tip engulfment on living macrophages, appropriate cell fixation procedure leaving the binding activity of FcγRs practically intact was elaborated. The recognition maps revealed prominent spots (microdomains) more or less homogeneously distributed on the macrophage surface with the sizes from 4 to 300 nm. Typical recognition image contained about â¼4% of large clusters (>200 nm), which were surrounded by a massive number (â¼50%) of small-size (4-30 nm) and the rest by middle-size (50, 150 nm) domains. These spots were detected from the decrease of oscillation amplitude during specific binding between Fc-coated tip and FcγRs on macrophage surfaces. In addition, the effect of osmotic swelling on the topographical landscape of macrophage surfaces and on the reorganization of FcγRs was investigated.