RESUMO
OBJECTIVE@#To explore the interaction between arginine functionalized hydroxyapatite (HAP/Arg) nanoparticles and endothelial cells, and to investigate mechanisms for endocytosis kinetics and endocytosis. @*METHODS@#Human umbilical vein endothelial cells (HUVECs) were selected as the research model.Cellular uptake of HAP/Arg nanoparticles were observed by laser scanning confocal microscopy.Average fluorescence intensity of cells after ingestion with different concentrations of HAP/Arg nanoparticles were determined by flow cytometer and atomic force microscopy. @*RESULTS@#The HAP/Arg nanoparticles with doped terbium existed in cytoplasm, and most of them distributed around the nucleus area after cellular uptake by HUVECs. Cellular uptake process of HAP/Arg nanoparticles in HUVECs was in a time and concentration dependent manner. 4 h and 50 mg/L was the best condition for uptake. HAP/Arg nanoparticles were easier to be up-taken into the cells than HAP nanoparticles without arginine functionalized. @*CONCLUSION@#HAP/Arg nanoparticles are internalized by HUVECs cells through an active transport and energy-dependent endocytosis process, and it is up-taken by cells mainly through caveolin-mediated endocytosis, but the clathrin-dependent endocytic pathway is also involved..
Assuntos
Humanos , Arginina , Farmacologia , Transporte Biológico Ativo , Fisiologia , Caveolinas , Fisiologia , Células Cultivadas , Clatrina , Fisiologia , Durapatita , Farmacocinética , Endocitose , Fisiologia , Células Endoteliais da Veia Umbilical Humana , Biologia Celular , Nanopartículas , MetabolismoRESUMO
This study investigated a nano drug delivery system built by one sort of modified trimethyl chitosan (TMC). The TMC was modified by cRGDyk, ligand of integrin receptor avβ3. Single factor screening was used to optimize the prescription in which the particle sizes of TMC nanoparticle (TMC NPs) and cRGDyk modified TMC nanoparticle (C-TMC NPs) were (240.3 ± 4.2) nm and (259.5 ± 3.3) nm. Electric potential of those two nanoparticles were (33.5 ± 0.8) mV and (25.7 ± 1.6) mV. Encapsulation efficiencies were (76.0 ± 2.2) % and (74.4 ± 2.0) %. Drug loading efficacies were (50.1 ± 2.1) % and (26.1 ± 1.0) %. Then the cellular uptake, uptake mechanism and transport efficacy of TMC NPs and C-TMC NPs were investigated using Caco-2 cell line. The uptake rate and accumulating drug transit dose of C-TMC NPs were 1.98 and 2.84 times higher than TMC NPs, separately. Mechanism investigations revealed that caveolae-mediated endocytosis, clathrin-mediated endocytosis and macropinocytosis were involved in the intercellular uptake of both TMC NPs and C-TMC NPs. What is more, free cRGDyk could remarkably inhibit the uptake of C-TMC NPs.
Assuntos
Humanos , Transporte Biológico , Células CACO-2 , Cavéolas , Quitosana , Química , Clatrina , Endocitose , Integrina alfaVbeta3 , Química , Nanopartículas , Tamanho da Partícula , PinocitoseRESUMO
BACKGROUND: The surgical treatment of advanced megaesophagus has no consensus, being esophagectomy the more commonly used method. Since it has high morbimortality - inconvenient for benign disease -, in recent years an alternative has been introduced: the esophageal mucosal resection. AIM: To compare early and late results of the two techniques evaluating the operative time, length of ICU stay; postoperative hospitalization; total hospitalization; intra- and postoperative complication rates; mortality; and long-term results. METHODS: Were evaluated retrospectively 40 charts, 23 esophagectomies and 17 mucosectomies. In assessing postoperative results, interviews were conducted by using a specific questionnaire. RESULTS: Comparing the means of esophagectomy and mucosal resection, respectively, the data were: 1) surgical time - 310.2 min and 279.7 min (p> 0.05); 2) length of stay in ICU - 5 days and 2.53 days (p <0.05); 3) total time of hospitalization - 24.25 days and 20.76 days (p> 0.05); 4) length of hospital stay after surgery - 19.05 days and 14.94 days (p> 0.05); 5) presence of intraoperative complications - 65% and 18% (p <0.05); 6) the presence of postoperative complications - 65% and 35% (p> 0.05). In the assessment of late postoperative score (range 0-10) esophagectomy (n = 5) obtained 8.8 points and 8.8 points also got mucosal resection (n = 5). CONCLUSIONS: Esophageal mucosal resection proved to be good alternative for surgical treatment of megaesophagus. It was advantageous in the immediate postoperative period by presenting a lower average time in operation, the total hospitalization, ICU staying and complications rate. In the late postoperative period, the result was excellent and good in both operations. .
RACIONAL: O tratamento cirúrgico do megaesôfago avançado não é consensual sendo mais comumente usada a esofagectomia. Por tratar-se de técnica que apresenta maior morbimortalidade e empregada em doença benigna, foi introduzida nos últimos anos, como alternativa, a mucosectomia esofágica. OBJETIVO: Comparar os resultados imediatos e tardios das duas técnicas avaliando-se os tempos operatório, de internação em UTI, de internação do pós-operatório, de internação total; taxas de complicações intra-operatórias e pós-operatórias; taxa de mortalidade; e resultados a longo prazo. MÉTODOS: Foram avaliados 40 prontuários, retrospectivamente, sendo 23 esofagectomias e 17 mucosectomias. Na avaliação dos resultados pós-operatórios, foram realizadas entrevistas, mediante uso de questionário específico. RESULTADOS: Comparando-se as médias da esofagectomia e mucosectomia, respectivamente, os dados foram: 1) tempo cirúrgico - 310,2 min e 279,7 min (p>0,05); 2) tempo de internação em UTI - 5 dias e 2,53 dias (p<0,05); 3) tempo de internação total - 24,25 dias e 20,76 dias (p>0,05); 4) tempo de internação após a operação - 19,05 dias e 14,94 dias (p>0,05); 5) presença de complicações intra-operatórias - 65% e 18% (p<0,05); 6) presença de complicações pós-operatórias imediatas - 65% e 35% (p>0,05). Na avaliação do escore pós-operatório tardio (escala 0-10) a esofagectomia (n=5) obteve 8,8 pontos e também 8,8 pontos obteve a mucosectomia (n=5). CONCLUSÕES: A mucosectomia esofágica mostrou-se boa alternativa no tratamento cirúrgico do megaesôfago avançado. Foi vantajosa no pós-operatório imediato por apresentar menor média de tempo na operação, na internação total, na UTI e no índice de complicações. No pós-operatório tardio, o resultado foi excelente e bom nas duas operações. .
Assuntos
Animais , Masculino , Camundongos , Metabolismo Energético , /metabolismo , Insulina/metabolismo , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Transdução de Sinais/fisiologia , Hipóxia/metabolismo , Células Cultivadas , Clatrina/metabolismo , /genética , Camundongos Transgênicos , Miócitos Cardíacos/citologia , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sarcolema/metabolismo , Sarcolema/ultraestruturaRESUMO
As the basic physiological function of synapses, vesicle cycling involves in many aspects of process. Among them, vesicle recycling is the basis of synaptic vesicle cycling. Studies show that clathrin mediated endocytosis is a major pathway of vesicle recycling, in which Dynamin plays an important role. Dynamin is a GTPases with molecular weight of 100 kD, which acts as "scissors" in the endocytosis, separating the clathrin coated pits from membrane. It has been found that Dynamin is associated with epilepsy, Alzheimer's disease, centronuclear myopathy, and several other neurological diseases. In this paper, we discussed the structure, function and regulation of Dynamin, and reviewed recent advance in the studies on Dynamin related diseases.
Assuntos
Humanos , Clatrina , Fisiologia , Invaginações Revestidas da Membrana Celular , Fisiologia , Dinaminas , Fisiologia , Endocitose , Sinapses , Fisiologia , Transmissão Sináptica , Vesículas Sinápticas , FisiologiaRESUMO
Porphyromonas gingivalis is one of the most important periodontal pathogens and has been to known to invade various types of cells, including endothelial cells. The present study investigated the mechanisms involved in the internalization of P. gingivalis in human umbilical vein endothelial cells (HUVEC). P. gingivalis internalization was reduced by clathrin and lipid raft inhibitors, as well as a siRNA knockdown of caveolin-1, a principal molecule of lipid raft-related caveolae. The internalization was also reduced by perturbation of actin rearrangement, while microtubule polymerization was not required. Furthermore, we found that Src kinases are critical for the internalization of P. gingivalis into HUVEC, while neither Rho family GTPases nor phosphatidylinositol 3-kinase are required. Taken together, this study indicated that P. gingivalis internalization into endothelial cells involves clathrin and lipid rafts and requires actin rearrangement associated with Src kinase activation.
Assuntos
Humanos , Actinas , Cavéolas , Caveolina 1 , Clatrina , Células Endoteliais , GTP Fosfo-Hidrolases , Células Endoteliais da Veia Umbilical Humana , Microtúbulos , Fosfatidilinositol 3-Quinase , Fosfotransferases , Polimerização , Polímeros , Porphyromonas gingivalis , RNA Interferente Pequeno , Quinases da Família srcRESUMO
Aanaplastic lymphoma kinase (ALK)-positive diffuse large B-cell lymphoma (DLBCL) is an unusual disease entity first reported in 1997 as DLBCL with expression of full-length ALK protein. The World Health Organization classification enlists the disease as a rare variant of DLBCL. Herein we describe two cases of ALK-positive DLBCL with cytomorphologic and molecular characteristics for the first time in Korea. The patients were 35-yr-old and 24-yr-old male patients. Immunohistochemical studies on the lymph nodes revealed large sized neoplastic cells with plasmablastic differentiation, which were negative for CD30 and positive for ALK with the characteristic granular staining in the cytoplasmic region. Extensive involvement of bone marrow was observed in both cases showing large, extremely atypical cells. Fluorescence in situ hybridization and molecular studies on the bone marrow aspirate specimens led to the detection of a clathrin (CLTC)/ALK rearrangement. Despite aggressive chemotherapy, the patients died 15 and 17 months after the diagnosis, indicating poor prognosis of the disease entity. This is the first report demonstrating the cytomorphologic findings of ALK-positive DLBCL cells on bone marrow aspirates.
Assuntos
Adulto , Humanos , Masculino , Medula Óssea/patologia , Clatrina/genética , Evolução Fatal , Fusão Gênica , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Linfoma Difuso de Grandes Células B/genética , Proteínas Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Kainic acid (KA) causes neurodegeneration, but no consensus has been reached concerning its mechanism. Nitric oxide may be a regulator of the mechanism. We identified differentially expressed genes in the hippocampus of mice treated with kainic acid, together with or without L-NAME, a nonselective nitric oxide synthase inhibitor, using a new differential display PCR method based on annealing control primers. Eight genes were identified, including clathrin light polypeptide, TATA element modulatory factor 1, neurexin III, ND4, ATPase, H+ transporting, V1 subunit E isoform 1, and N-myc downstream regulated gene 2. Although the functions of these genes and their products remain to be determined, their identification provides insight into the molecular mechanism(s) involved in KA-induced neuronal cell death in the hippocampal CA3 area.
Assuntos
Animais , Camundongos , Morte Celular , Clatrina , Consenso , Hipocampo , Ácido Caínico , Modelos Animais , Neurônios , NG-Nitroarginina Metil Éster , Óxido Nítrico Sintase , Óxido Nítrico , Reação em Cadeia da Polimerase , ATPases Translocadoras de PrótonsRESUMO
<p><b>OBJECTIVE</b>To study the characteristics of a novel human testis-specific gene, HSD-9, and its encoding protein.</p><p><b>METHODS</b>HSD-9 was a novel gene from a human germ cells-specific ESTs library established in our laboratory. We used an electronic cloning method to obtain HSD-9 gene, and analyzed the characteristics of this novel gene and encoding product by bioinformatics methods, detected its expressing profile using a Northern blot assay, prepared specific rabbit polyclonal antibodies against HSD-9 protein, observed the localization of this protein in the germ cells and some somatic cells with confocal microscopy.</p><p><b>RESULTS</b>HSD-9 was expressed in human testes, and its rat homolog was found in the varying germ cells. HSD-9 protein could partly be colocalized with clathrin.</p><p><b>CONCLUSIONS</b>HSD-9 is specific in human testes, and the expression pattern of its encoding product is similar to those of some endocytosis proteins. It is speculated that HSD-9 protein may function in the endocytosis.</p>
Assuntos
Animais , Humanos , Masculino , Coelhos , Ratos , Sequência de Aminoácidos , Sequência de Bases , Clatrina , Metabolismo , Proteínas de Membrana , Genética , Dados de Sequência Molecular , Especificidade de Órgãos , Testículo , MetabolismoRESUMO
Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the protein and lipid. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). In order to better understand a possible role of post-translational modification of CALM (clathrin assembly protein lymphoid myeloid), the homologue of AP180, in the assembly of CCVs, CALM was expressed in the cell-free reticulocyte translation system that is capable of carrying out post-translational modification. The apparent molecular weight of the expressed recombinant CALM was estimated as 105 kD. Alkaline phosphatase treatment of CALM resulted in a mobility shift on SDS-PAGE. We found that CALM was associated with the proteins harboring SH3 domain, promote assembly of clathrin triskelia into clathrin cage and bound to the preformed clathrin cage. CALM was also proteolyzed by caspase 3 and calpain but not by caspase 8. These results indicated that the post-translationally modified CALM, expressed in the eukaryotic cell-free reticulocyte translation system was able to mediate the assembly of clathrin and the coated-vesicle formation.
Assuntos
Bovinos , Fosfatase Alcalina/farmacologia , Animais , Encéfalo/metabolismo , Calpaína/metabolismo , Proteínas de Transporte/química , Caspases/metabolismo , Sistema Livre de Células , Clatrina/química , Eletroforese em Gel de Poliacrilamida , Glutationa Transferase/metabolismo , Lipídeos/química , Proteínas de Membrana/química , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes/química , Reticulócitos/metabolismo , Biossíntese de Proteínas , Domínios de Homologia de srcRESUMO
The most efficient means of protein internalization from the membrane are through clathrin-coated pits, which concentrate protein interactions with the clathrin-associated assembly protein complex AP-2 and internalization signals in the cytoplasmic domain of transmembrane proteins. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). Due to a difficulty of isolating clathrin molecules from their complex or assembly state in the cells, most of the studies were carried out with recombinant clathrin proteins, which may present different conformation and structural variation. In this study, we have developed an efficient method of isolating the native clathrin assembly protein lymphoid myeloid (CALM) from the bovine brain that is enriched with clathrin and clathrin associated proteins and characterized by their sensitivity to proteases and it's ability to form CCV. The purified CALM has molecular weight of approximately 100,000 dalton on SDS-PAGE, which is consistent with the result of in vitro translation. The purified CALM protein could promote the assembly of clathrin triskelia into clathrin cage, and cleaved CALM proteolysed by caspase 3 and calpain could not promote them. In this respect, our data support a model in which CALM functions like AP180 as a monomeric clathrin assembly protein and might take part in apoptotic process in neuronal cells.
Assuntos
Bovinos , Proteínas Adaptadoras de Transporte Vesicular , Animais , Química Encefálica , Calpaína/metabolismo , Proteínas de Transporte , Caspases/metabolismo , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Hidrólise , Proteínas de Membrana , Peso Molecular , Proteínas do Tecido Nervoso/química , Neurônios/química , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/químicaRESUMO
The most efficient means of protein internalization from the membrane are through clathrin-coated pits, which concentrate protein interactions with the clathrin-associated assembly protein complex AP-2 and internalization signals in the cytoplasmic domain of transmembrane proteins. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). Due to a difficulty of isolating clathrin molecules from their complex or assembly state in the cells, most of the studies were carried out with recombinant clathrin proteins, which may present different conformation and structural variation. In this study, we have developed an efficient method of isolating the native clathrin assembly protein lymphoid myeloid (CALM) from the bovine brain that is enriched with clathrin and clathrin associated proteins and characterized by their sensitivity to proteases and it's ability to form CCV. The purified CALM has molecular weight of approximately 100,000 dalton on SDS-PAGE, which is consistent with the result of in vitro translation. The purified CALM protein could promote the assembly of clathrin triskelia into clathrin cage, and cleaved CALM proteolysed by caspase 3 and calpain could not promote them. In this respect, our data support a model in which CALM functions like AP180 as a monomeric clathrin assembly protein and might take part in apoptotic process in neuronal cells.
Assuntos
Bovinos , Proteínas Adaptadoras de Transporte Vesicular , Animais , Química Encefálica , Calpaína/metabolismo , Proteínas de Transporte , Caspases/metabolismo , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Hidrólise , Proteínas de Membrana , Peso Molecular , Proteínas do Tecido Nervoso/química , Neurônios/química , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/químicaRESUMO
Recently, the gene encoding clathrin assembly protein of lymphoid myeloid leukemia (CALM), which is homologous to the AP180, was cloned from rat brain, and its expression differential to AP180 was reported (Kim and Lee, 1999). This gene product promotes the polymerization of clathrin into clathrin cage and found to be a regulator in membrane trafficking between intracellular compartments in eukaryotic cells (Kim et al., 2000). In this study, we have purified the CALM protein from clathrin-coated vesicles of rat liver using the monoclonal antibody against the recombinant N-terminal region of the CALM. The coated proteins extracted from the coated vesicle fraction was further purified by multi-step procedures involving gel-filtration and ion-exchange chromatography and SDS-PAGE. The purified protein with an apparent molecular weight of 100 kD promoted the assembly of clathrin triskelia into clathrin cage. In this respect the CALM protein bears a functional resemblance to the AP180 that has been previously described.
Assuntos
Ratos , Animais , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/química , Fígado/química , Proteínas do Tecido Nervoso/isolamento & purificação , Fosfoproteínas/isolamento & purificaçãoAssuntos
Animais , Proteínas de Membrana/classificação , Vesículas Revestidas/metabolismo , Transporte Biológico , Clatrina , Proteína Coatomer , Guanosina Trifosfato , Substâncias Macromoleculares , Modelos Biológicos , Proteínas de Membrana/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Virais/metabolismo , Saccharomyces cerevisiaeAssuntos
Humanos , Animais , Clatrina , Proteínas de Membrana/fisiologia , Vesículas Revestidas/metabolismo , Subunidades alfa do Complexo de Proteínas Adaptadoras , Subunidades beta do Complexo de Proteínas Adaptadoras , Subunidades gama do Complexo de Proteínas Adaptadoras , Proteínas Adaptadoras de Transporte Vesicular , Fosfatase Ácida/química , Fosfatase Ácida/fisiologia , Lisossomos , Substâncias Macromoleculares , Conformação Proteica , Proteínas de Membrana/química , Tirosina , Vesículas Revestidas/ultraestruturaRESUMO
En la face de maduración o condensación del complejo de Golgi de la espermátide de cobayo, se presentan las membranas de las cisternas densas y de las vesículas de condensación con características especiales que las diferencian del resto de las membranas del Golgi. Las membranas de la fase de condensación son más gruesas, están en su mayor parte cubiertas por una trama poligonal del tipo característico de la clathrina y tratadas con la técnica de la digitonina forman los típicos rulos y agujas del complejo digitonina-colesterol. Esta observación revela que ambos componentes - clathrina y colesterol - pueden coincidir en la misma membrana, se concentran en la cara interna del Golgi y por fusión de membranas se transfieren a la membrana externa del acrosoma