RESUMO
Regulation of fatty acid uptake, lipid production and storage, and metabolism of lipid droplets (LDs), is closely related to lipid homeostasis, adipocyte hypertrophy and obesity. We report here that stomatin, a major constituent of lipid raft, participates in adipogenesis and adipocyte maturation by modulating related signaling pathways. In adipocyte-like cells, increased stomatin promotes LD growth or enlargements by facilitating LD-LD fusion. It also promotes fatty acid uptake from extracellular environment by recruiting effector molecules, such as FAT/CD36 translocase, to lipid rafts to promote internalization of fatty acids. Stomatin transgenic mice fed with high-fat diet exhibit obesity, insulin resistance and hepatic impairments; however, such phenotypes are not seen in transgenic animals fed with regular diet. Inhibitions of stomatin by gene knockdown or OB-1 inhibit adipogenic differentiation and LD growth through downregulation of PPARγ pathway. Effects of stomatin on PPARγ involves ERK signaling; however, an alternate pathway may also exist.
Assuntos
Adipogenia , Gotículas Lipídicas , Adipogenia/genética , Animais , Antígenos CD36/genética , Antígenos CD36/metabolismo , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Sistema de Sinalização das MAP Quinases , Camundongos , Obesidade/genética , Obesidade/metabolismo , PPAR gama/genética , PPAR gama/metabolismoRESUMO
Rapid bacterial antibiotic susceptibility test (AST) and minimum inhibitory concentration (MIC) measurement are important to help reduce the widespread misuse of antibiotics and alleviate the growing drug-resistance problem. We discovered that, when a susceptible strain of Staphylococcus aureus or Escherichia coli is exposed to an antibiotic, the intensity of specific biomarkers in its surface-enhanced Raman scattering (SERS) spectra drops evidently in two hours. The discovery has been exploited for rapid AST and MIC determination of methicillin-susceptible S. aureus and wild-type E. coli as well as clinical isolates. The results obtained by this SERS-AST method were consistent with that by the standard incubation-based method, indicating its high potential to supplement or replace existing time-consuming methods and help mitigate the challenge of drug resistance in clinical microbiology.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Análise Espectral Raman/métodos , Staphylococcus aureus/efeitos dos fármacos , Biomarcadores , Células Cultivadas , Escherichia coli/metabolismo , Staphylococcus aureus/metabolismoRESUMO
Our previous studies showed that colorectal tumor has high interleukin-4 receptor α (IL-4Rα) expression, whereas adjacent normal tissue has low or no IL-4Rα expression. We also observed that human atherosclerotic plaque-specific peptide-1 (AP1) can specifically target to IL-4Rα. In this study, we investigated the therapeutic efficacy and systemic toxicity of AP1-conjuagted liposomal doxorubicin. AP1 bound more strongly to and was more efficiently internalized into IL-4Rα-overexpressing CT26 cells than CT26 control cells. Selective cytotoxicity experiment revealed that AP1-conjugated liposomal doxorubicin preferentially killed IL-4Rα-overexpressing CT26 cells. AP1-conjugated liposomal doxorubicin administered intravenously into mice produced significant inhibition of tumor growth and showed decreased cardiotoxicity of doxorubicin. These results indicated that AP1-conjugated liposomal doxorubicin has a potent and selective anticancer potential against IL-4Rα-overexpressing colorectal cancer cells, thus providing a model for targeted anticancer therapy.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Doxorrubicina/administração & dosagem , Peptídeos/administração & dosagem , Receptores de Interleucina-4/metabolismo , Animais , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Doxorrubicina/metabolismo , Doxorrubicina/toxicidade , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Lipossomos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Peptídeos/metabolismo , Peptídeos/toxicidade , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacosRESUMO
The addition of copper chlorophyll and its derivatives (Cu-Chl) to vegetable oils to disguise them as more expensive oils, such as virgin olive oils, would not only create public confusion, but also disturb the olive oil market. Given that existing detection methods of Ch-Chl in oils, such as LC-MS are costly and time consuming, it is imperative to develop economical and fast analytical techniques to provide information quickly. This paper demonstrates a rapid analytical method based on surface-enhanced Raman spectroscopy (SERS) to detect Cu-Chl in vegetable oils; the spectroscopic markers of Cu-Chl are presented and a detection limit of 5 mg kg(-1) is demonstrated. The analysis of a series of commercial vegetable oils is undertaken with this method and the results verified by a government agency. This study shows that a SERS-based assessment method holds high potential for quickly pinpointing the addition of minute amounts of Cu-Chl in vegetable oils.
Assuntos
Clorofilídeos/análise , Azeite de Oliva/química , Óleos de Plantas/química , Óleo de Soja/química , Análise Espectral Raman , Análise de Alimentos , Contaminação de Alimentos/análise , Limite de Detecção , Reprodutibilidade dos Testes , Óleo de GirassolRESUMO
In this study, we developed the cancer treatment through the combination of chemotherapy and thermotherapy using doxorubicin-loaded magnetic liposomes. The citric acid-coated magnetic nanoparticles (CAMNP, ca. 10 nm) and doxorubicin were encapsulated into the liposome (HSPC/DSPE/cholesterol = 12.5:1:8.25) by rotary evaporation and ultrasonication process. The resultant magnetic liposomes (ca. 90 to 130 nm) were subject to characterization including transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray diffraction (XRD), zeta potential, Fourier transform infrared (FTIR) spectrophotometer, and fluorescence microscope. In vitro cytotoxicity of the drug carrier platform was investigated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using L-929 cells, as the mammalian cell model. In vitro cytotoxicity and hyperthermia (inductive heating) studies were evaluated against colorectal cancer (CT-26 cells) with high-frequency magnetic field (HFMF) exposure. MTT assay revealed that these drug carriers exhibited no cytotoxicity against L-929 cells, suggesting excellent biocompatibility. When the magnetic liposomes with 1 µM doxorubicin was used to treat CT-26 cells in combination with HFMF exposure, approximately 56% cells were killed and found to be more effective than either hyperthermia or chemotherapy treatment individually. Therefore, these results show that the synergistic effects between chemotherapy (drug-controlled release) and hyperthermia increase the capability to kill cancer cells.
RESUMO
Radial glial progenitor cells exhibit bidirectional cell cycle-dependent nuclear oscillations. The purpose and underlying mechanism of this unusual 'interkinetic nuclear migration' are poorly understood. We investigated the basis for this behavior by live imaging of nuclei, centrosomes and microtubules in embryonic rat brain slices, coupled with the use of RNA interference (RNAi) and the myosin inhibitor blebbistatin. We found that nuclei migrated independent of centrosomes and unidirectionally away from or toward the ventricular surface along microtubules, which were uniformly oriented from the ventricular surface to the pial surface of the brain. RNAi directed against cytoplasmic dynein specifically inhibited nuclear movement toward the apical surface. An RNAi screen of kinesin genes identified Kif1a, a member of the kinesin-3 family, as the motor for basally directed nuclear movement. These observations provide direct evidence that kinesins are involved in nuclear migration and neurogenesis and suggest that a cell cycle-dependent switch between distinct microtubule motors drives interkinetic nuclear migration.
Assuntos
Movimento Celular/fisiologia , Núcleo Celular/fisiologia , Dineínas do Citoplasma/fisiologia , Células-Tronco Embrionárias/fisiologia , Cinesinas/fisiologia , Células-Tronco Neurais/fisiologia , Animais , Ciclo Celular/fisiologia , Células-Tronco Embrionárias/citologia , Cinese/fisiologia , Microtúbulos/fisiologia , Células-Tronco Neurais/citologia , Células PC12 , RatosRESUMO
Asymmetric divisions of radial glia progenitors produce self-renewing radial glia and differentiating cells simultaneously in the ventricular zone (VZ) of the developing neocortex. Whereas differentiating cells leave the VZ to constitute the future neocortex, renewing radial glia progenitors stay in the VZ for subsequent divisions. The differential behaviour of progenitors and their differentiating progeny is essential for neocortical development; however, the mechanisms that ensure these behavioural differences are unclear. Here we show that asymmetric centrosome inheritance regulates the differential behaviour of renewing progenitors and their differentiating progeny in the embryonic mouse neocortex. Centrosome duplication in dividing radial glia progenitors generates a pair of centrosomes with differently aged mother centrioles. During peak phases of neurogenesis, the centrosome retaining the old mother centriole stays in the VZ and is preferentially inherited by radial glia progenitors, whereas the centrosome containing the new mother centriole mostly leaves the VZ and is largely associated with differentiating cells. Removal of ninein, a mature centriole-specific protein, disrupts the asymmetric segregation and inheritance of the centrosome and causes premature depletion of progenitors from the VZ. These results indicate that preferential inheritance of the centrosome with the mature older mother centriole is required for maintaining radial glia progenitors in the developing mammalian neocortex.
Assuntos
Divisão Celular , Linhagem da Célula , Centrossomo/fisiologia , Neocórtex/citologia , Neurônios/citologia , Células-Tronco/citologia , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Senescência Celular/fisiologia , Centríolos/fisiologia , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Humanos , Camundongos , Neocórtex/embriologia , Neurogênese/fisiologia , Neuroglia/citologia , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologiaRESUMO
To deliver non-permeable molecules into cells, one can utilize protocols such as microinjection, electroporation, liposome-mediated transfection or virus-mediated transfection. However, each method has its own limitations. Here we have developed a new molecular delivery technique where live cells or tissues are bombarded with highly accelerated molecules directly and without the need to conjugate the molecules onto carrier particles, which is essential in conventional "gene gun" experiments. Gene bombardments can be applied to well-differentiated cells, primary cultured cells/neurons or tissue explants, all of which are notoriously difficult to transfect. Exogenously made proteins and even bacteria can be effectively introduced into cells where they can execute their function or replicate. Our experimental results and physical model support the notion that accelerated chemicals, proteins, or microorganisms carry enough momentum to penetrate the plasma membrane. The bombardment process is associated with a transient (approximately 10 min) increase in cell permeability, but such membrane leakage has a minimal adverse effect on cell survival.
Assuntos
Bactérias , Biolística/métodos , Sistemas de Liberação de Medicamentos/métodos , Animais , Técnicas Bacteriológicas , Células CHO , Linhagem Celular , Permeabilidade da Membrana Celular , Cricetinae , Cricetulus , Proteínas do Citoesqueleto/administração & dosagem , Proteínas do Citoesqueleto/química , DNA Recombinante/administração & dosagem , DNA Recombinante/genética , Escherichia coli , Células HeLa , Humanos , Modelos Biológicos , Peso Molecular , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , TransfecçãoRESUMO
Polarized epithelia, such as hepatocytes, target their integral membrane proteins to specific apical or basolateral membrane domains during or after biogenesis. The roles played by protein glycosylation in this sorting process remain controversial. We report here that deglycosylation treatments in well-polarized hepatic cells by deglycosylation drugs, or by site-directed mutagenesis of the N-linked-glycosylation residues, all cause the Na+/K+-ATPase beta-subunit to traffic from the native basolateral to the apical/canalicular domain. Deglycosylated beta-subunits are still able to bind and therefore transport the catalytic alpha-subunits to the aberrant apical location. Such apical targeting is mediated via the indirect transcytosis pathway. Cells containing apical Na+/K+-ATPase appear to be defective in maintaining the ionic gradient across the plasma membrane and in executing hepatic activities that are dependent upon the ionic homeostasis such as canalicular excretion.
Assuntos
Hepatócitos/metabolismo , Subunidades Proteicas/metabolismo , Transporte Proteico/fisiologia , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Antibacterianos/farmacologia , Linhagem Celular , Polaridade Celular , Glicosilação , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Homeostase , Humanos , Mutagênese Sítio-Dirigida , Subunidades Proteicas/química , Subunidades Proteicas/genética , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Tunicamicina/farmacologiaRESUMO
PICK1 binds to protein kinase Calpha (PKCalpha) through the carboxylate-binding loop in its PDZ (PSD95/Disc-large/ZO-1) domain and the C terminus of PKCalpha. We have previously shown that PICK1 modulates the catalytic activity of PKC selectively toward the antiproliferative gene TIS21. To investigate whether PICK1 plays a role in targeting activated PKCalpha to a particular intracellular compartment in addition to regulating PKC activity, we examine the localization of PICK1 and PKCalpha in response to various stimuli. Double staining with organelle markers and anti-rPICK1 antibodies reveals that PICK1 is associated with mitochondria but not with endoplasmic reticulum or Golgi in NIH 3T3 cells. Deletion of the PDZ domain impairs the mitochondria localization of PICK1, whereas mutations in the carboxylate-binding loop do not have an effect, suggesting that PICK1 can bind PKCalpha and mitochondria simultaneously. Upon serum stimulation, PICK1 translocates and displays a dense ring-like structure around the nucleus, where it still associates with mitochondria. A substantial portion of PKCalpha is concomitantly found in the condense perinuclear region. The C terminal-deleted PKCalpha fails to translocate and remains a diffuse cytoplasmic distribution, indicating that a direct interaction between PICK1 and PKCalpha is required for PKCalpha anchoring to mitochondria. 12-O-Tetradecanoylphorbol-13-acetate stimulation, in contrast, causes translocation of PKCalpha to the plasma membrane, whereas the majority of PICK1 remains in a cytoplasmic punctate pattern. Deletion at the C terminus of PKCalpha has no effect on 12-O-tetradecanoylphorbol-13-acetate-induced translocation. These findings indicate a previously unidentified role for PICK1 in anchoring PKCalpha to mitochondria in a ligand-specific manner.