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1.
Clin Oral Implants Res ; 35(6): 630-640, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38567929

RESUMO

OBJECTIVES: This RCT aimed to compare zirconia and titanium dental implants in the maxillary premolar region. The comparison was based on marginal bone level (MBL) changes, clinical parameters, aesthetic outcomes, and patient related outcome measures (PROMs) 1 year after prosthetic loading. MATERIALS AND METHODS: Fifty patients were randomly assigned to receive either a zirconia (ZrO2, n = 25) implant or a titanium (Ti, n = 25) bone-level implant. Implants were provided with a lithium disilicate crown 3 months after placement. Follow-up was at 1 month and after 1 year. The primary outcome pertained to changes in MBL. Reported secondary outcomes consisted of implant survival, peri-implant tissue health, aesthetics, and PROMs. RESULTS: Mean MBL change after 1 year was 0.01 mm (SD = 0.45; min = 0.72, max = 0.86) for ZrO2 and -0.09 mm (SD = 0.34; min = 0.53, max = -1.06) for Ti (p = .439). Scores for the other clinical outcome parameters and PROMs were generally favorable, with no significant differences. However, significant differences were found for the aesthetic outcomes regarding two criteria: (a) level of facial mucosa (p = .022), in favor of Ti, and (b) root convexity/soft tissue color and texture (p = .005) in favor of ZrO2. CONCLUSION AND CLINICAL IMPLICATIONS: The ZrO2 and Ti implant types used in this study, replacing a single missing maxillary premolar, show a comparable outcome in terms of MBL change after 1 year. Clinical and aesthetic parameters, as well as PROMs, are favorable and similar between both implant types after 1 year of prosthetic loading. These short-term study results suggest that both are suitable for clinical use.


Assuntos
Implantes Dentários para Um Único Dente , Maxila , Titânio , Zircônio , Humanos , Feminino , Masculino , Pessoa de Meia-Idade , Maxila/cirurgia , Adulto , Estética Dentária , Idoso , Resultado do Tratamento , Coroas , Implantação Dentária Endóssea/métodos , Prótese Dentária Fixada por Implante , Dente Pré-Molar
2.
J Oral Rehabil ; 44(12): 988-995, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28856707

RESUMO

The aim of this study was to assess the 5-year treatment outcome of maxillary implant-retained overdentures opposed by natural antagonistic teeth. Fifty consecutive patients received maxillary overdentures supported by six dental implants. Implants were placed in the anterior region, if enough bone was present (n = 25 patients) Implant were placed in the posterior region if implant placement in the anterior region was not possible (n = 25 patients). Variables assessed included survival of implants, condition of hard and soft peri-implant tissues and patients' satisfaction. The five-year implant survival rate was 97·0% and 99·3%, and mean radiographic bone loss was 0·23 and 0·69 mm in the anterior and posterior group, respectively. Median scores for plaque, calculus, gingiva, bleeding and mean scores for pocket probing depth were low and stayed low. Patients' satisfaction after treatment was high in both groups. Within the limits of this 5-year study, it is concluded that six dental implants (placed in the anterior or posterior region) connected with a bar and opposed to natural antagonistic teeth result in acceptable results for clinical parameters and good outcomes for marginal bone level changes and patient satisfaction.


Assuntos
Prótese Dentária Fixada por Implante , Revestimento de Dentadura , Maxila/cirurgia , Boca Edêntula/cirurgia , Adulto , Idoso , Implantes Dentários , Feminino , Humanos , Masculino , Maxila/diagnóstico por imagem , Maxila/patologia , Pessoa de Meia-Idade , Estudos Prospectivos , Radiografia Panorâmica
3.
Ned Tijdschr Tandheelkd ; 118(11): 577-82, 2011 Nov.
Artigo em Holandês | MEDLINE | ID: mdl-22235521

RESUMO

An implant-supported overdenture is a good alternative treatment to a conventional denture for patients with complaints about the retention and stability of their removable complete denture. These complaints more often have to do with the mandibular than the maxillary denture. Implant-supported overdentures offer better results in the mandible than in the maxilla. In cases of insujficient bone volume in the maxilla for inserting implants, maxillary sinus floor elevation using an autogenous bone graft from the oral cavity or the iliac crest may be carried out. Treatment of the edentulous maxilla by inserting 6 implants followed by manufacturing a bar-clip mesostructure and an implant-supported overdenture is the most successful, followed closely by the treatment option of inserting 4 implants and fabricating a similar mesostructure and overdenture. Aftercare by routine preventive examinations is required.


Assuntos
Retenção em Prótese Dentária , Prótese Dentária Fixada por Implante , Arcada Edêntula/reabilitação , Revestimento de Dentadura , Humanos , Maxila , Satisfação do Paciente , Resultado do Tratamento
4.
Ned Tijdschr Tandheelkd ; 115(11): 599-604, 2008 Nov.
Artigo em Holandês | MEDLINE | ID: mdl-19058505

RESUMO

Patients with an edentulous maxilla can experience problems with a full upper denture. The most common problems are a lack of retention and the stability of the denture, but also other factors, such as an extreme gagging reflex, influence satisfaction. Attachment of a prosthesis on dental implants is a reliable solution to solve or diminish the above mentioned problems. The choice of the kind of superstructure, a removable overdenture or a fixed prosthesis, depends on a variety of factors, such as degree of resorption of the maxilla, cleaning possibilities, patients'wishes and financial possibilities


Assuntos
Planejamento de Prótese Dentária , Prótese Dentária Fixada por Implante/métodos , Arcada Edêntula/reabilitação , Maxila/cirurgia , Satisfação do Paciente , Prótese Dentária Fixada por Implante/economia , Planejamento de Dentadura , Retenção de Dentadura , Revestimento de Dentadura , Humanos , Resultado do Tratamento
5.
Ned Tijdschr Tandheelkd ; 111(10): 388-91, 2004 Oct.
Artigo em Holandês | MEDLINE | ID: mdl-15553367

RESUMO

Aim of this study is the evaluation of a 3-weeks course on dental implants presented in a computer assisted learning program. Evaluation variables are study result, student satisfaction and the use of the program. Fourth grade dental students were divided in a group who received traditional education and a group who received the course by means of an interactive computerprogram. At the end of the experiment there is no difference in study result between the two groups. Students say that the computer assisted learning program is a useful tool in studying the course, but use the program more as a device to obtain information than as a tool to have interactive discussion.


Assuntos
Instrução por Computador , Implantação Dentária/educação , Educação em Odontologia , Estudantes de Odontologia , Educação em Odontologia/métodos , Educação em Odontologia/estatística & dados numéricos , Avaliação Educacional , Humanos , Projetos Piloto
6.
Proc Natl Acad Sci U S A ; 100(23): 13332-7, 2003 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-14597718

RESUMO

Endosomes are major sorting stations in the endocytic route that send proteins and lipids to multiple destinations in the cell, including the cell surface, Golgi complex, and lysosomes. They have an intricate architecture of internal membrane structures enclosed by an outer membrane. Recycling proteins remain on the outer membrane, whereas proteins that are destined for degradation in the lysosome are sorted to the interior. Recently, a retrograde pathway was discovered whereby molecules, like MHC class II of the immune system, return from the internal structures to the outer membrane, allowing their further transport to the cell surface for T cell activation. Whether this return involves back fusion of free vesicles with the outer membrane, or occurs via the continuity of the two membrane domains, is an unanswered question. By electron tomography of cryo-immobilized cells we now demonstrate that, in multivesicular endosomes of B-lymphocytes and dendritic cells, the inner membranes are free vesicles. Hence, protein transport from inner to outer membranes cannot occur laterally in the plane of the membrane, but requires fusion between the two membrane domains. This implies the existence of an intracellular machinery that mediates fusion between the exoplasmic leaflets of the membranes involved, which is opposite to regular intracellular fusion between cytoplasmic leaflets. In addition, our 3D reconstructions reveal the presence of clathrin-coated areas at the cytoplasmic face of the outer membrane, known to participate in protein sorting to the endosomal interior. Interestingly, profiles reminiscent of inward budding vesicles were often in close proximity to the coats.


Assuntos
Endossomos/fisiologia , Endossomos/ultraestrutura , Fusão de Membrana/fisiologia , Animais , Linfócitos B/citologia , Linhagem Celular , Linhagem Celular Transformada , Clatrina/metabolismo , Citoplasma/metabolismo , Células Dendríticas/metabolismo , Endossomos/metabolismo , Congelamento , Humanos , Microscopia Imunoeletrônica , Ratos , Linfócitos T/citologia
7.
J Thromb Haemost ; 1(6): 1161-73, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12871315

RESUMO

The molecular mechanism that causes non-adhesive, discoid platelets to transform into sticky dendritic bodies that form blood clumps is a complex series of events. Recently it has become clear that lipid microdomains--also known as rafts--play a crucial role in this process. We have used a non-cytolytic derivative of perfringolysin-O, a cholesterol binding cytolysin, that binds selectively to cholesterol-rich membrane domains, combined with confocal- and immunoelectron microscopy to visualize cholesterol-raft dynamics during platelet adhesion. In resting platelets cholesterol was uniformly distributed on the cell surface and confined to distinct intracellular compartments (i.e. multivesicular bodies, dense granules, and the internal membranes of alpha-granules). Upon interaction with fibrinogen, cholesterol accumulated at the tips of filopodia and at the leading edge of spreading cells. Stimulation with thrombin receptor activating peptide (TRAP) resulted in a similar redistribution of cholesterol towards filopodia. The adhesion-dependent raft aggregation was accompanied by concentration of the tyrosine kinase c-Src and the tetraspanin CD63 in these domains, whereas glycoprotein Ib (GPIb) was not selectively targeted to the raft clusters. c-Src, the tetraspanin CD63, and GPIb were recovered in biochemically isolated low-density membrane fractions. Disruption of rafts by depleting membrane cholesterol had no effect on platelet shape change but inhibited platelet spreading on fibrinogen and TRAP-induced aggregation. Our results demonstrate that cholesterol rafts in platelets are dynamic entities in the membrane that co-cluster with the tyrosine kinase c-Src and the costimulatory molecule CD63 in specialized domains at the cell surface, thereby providing a possible mechanism in functioning as signaling centres.


Assuntos
Antígenos CD/metabolismo , Plaquetas/ultraestrutura , Microdomínios da Membrana/fisiologia , Fosfotransferases/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pseudópodes/química , beta-Ciclodextrinas , Plaquetas/química , Plaquetas/fisiologia , Proteína Tirosina Quinase CSK , Tamanho Celular , Colesterol/metabolismo , Colesterol/fisiologia , Ciclodextrinas/farmacologia , Fibrinogênio , Humanos , Imuno-Histoquímica , Microdomínios da Membrana/química , Fosforilação , Ativação Plaquetária , Transporte Proteico , Proteínas Tirosina Quinases , Receptores de Trombina , Tetraspanina 30 , Quinases da Família src
8.
Traffic ; 4(4): 222-31, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12694561

RESUMO

We employed our recently developed immuno-electron microscopic method (W. Möbius, Y. Ohno-Iwashita, E. G. van Donselaar, V. M. Oorschot, Y. Shimada, T. Fujimoto, H. F. Heijnen, H. J. Geuze and J. W. Slot, J Histochem Cytochem 2002; 50: 43-55) to analyze the distribution of cholesterol in the endocytic pathway of human B lymphocytes. We could distinguish 6 categories of endocytic compartments on the basis of morphology, BSA gold uptake kinetics and organelle marker analysis. Of all cholesterol detected in the endocytic pathway, we found 20% in the recycling tubulo-vesicles and 63% present in two types of multivesicular bodies. In the multivesicular bodies, most of the cholesterol was contained in the internal membrane vesicles, the precursors of exosomes secreted by B cells. Cholesterol was almost absent from lysosomes, that contained the bulk of the lipid bis(monoacylglycero)phosphate, also termed lysobisphosphatidic acid. Thus, cholesterol displays a highly differential distribution in the various membrane domains of the endocytic pathway.


Assuntos
Colesterol/metabolismo , Endocitose , Linhagem Celular Transformada , Endossomos/metabolismo , Endossomos/ultraestrutura , Ouro/metabolismo , Humanos , Cinética , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Microscopia Imunoeletrônica , Soroalbumina Bovina/metabolismo
9.
Traffic ; 2(11): 831-8, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11733050

RESUMO

By using quantitative immuno-electron microscopy of two-sided labeled resin sections of rat exocrine pancreatic cells, we have established the relative concentrations of the secretory proteins amylase and chymotrypsinogen in the compartments of the secretory pathway. Their total concentration over the entire pathway was approximately 11 and approximately 460 times, respectively. Both proteins exhibited their largest increase in concentration between the endoplasmic reticulum and cis-Golgi, where they were concentrated 3-4 and 50-70 times, respectively. Over the further pathway, increases in concentration were moderate, albeit two times higher for chymotrypsinogen than for amylase. From trans-Golgi to secretory granules, where the main secretory protein concentration is often thought to occur, relatively small concentration increases were observed. Additional observations on a third secretory protein, procarboxypeptidase A, showed a concentration profile very similar to chymotrypsinogen. The relatively high concentration of amylase in the early compartments of the secretory route is consistent with its exceptionally slow intracellular transport. Our data demonstrate that secretory proteins undergo their main concentration between the endoplasmic reticulum and cis-Golgi, where we have previously found concentration activity associated with vesicular tubular clusters (Martínez-Menárguez JA, Geuze HJ, Slot JW, Klumperman J. Cell 1999; 98: 81-90).


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Pâncreas/metabolismo , Animais , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Masculino , Microscopia Imunoeletrônica , Pâncreas/citologia , Pâncreas/ultraestrutura , Transporte Proteico , Ratos , Ratos Wistar
10.
J Cell Biol ; 155(7): 1213-24, 2001 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-11748250

RESUMO

A cisternal progression mode of intra-Golgi transport requires that Golgi resident proteins recycle by peri-Golgi vesicles, whereas the alternative model of vesicular transport predicts anterograde cargo proteins to be present in such vesicles. We have used quantitative immuno-EM on NRK cells to distinguish peri-Golgi vesicles from other vesicles in the Golgi region. We found significant levels of the Golgi resident enzyme mannosidase II and the transport machinery proteins giantin, KDEL-receptor, and rBet1 in coatomer protein I-coated cisternal rims and peri-Golgi vesicles. By contrast, when cells expressed vesicular stomatitis virus protein G this anterograde marker was largely absent from the peri-Golgi vesicles. These data suggest a role of peri-Golgi vesicles in recycling of Golgi residents, rather than an important role in anterograde transport.


Assuntos
Ciclo Celular/fisiologia , Complexo de Golgi/fisiologia , Glicoproteínas de Membrana , Transporte Proteico , Animais , Autoantígenos/metabolismo , Linhagem Celular , Complexo I de Proteína do Envoltório , Complexo de Golgi/ultraestrutura , Proteínas da Matriz do Complexo de Golgi , Proteínas de Fluorescência Verde , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Rim , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Imunoeletrônica , Ratos , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Transfecção , Vírus da Estomatite Vesicular Indiana/fisiologia , Proteínas do Envelope Viral/metabolismo
11.
Am J Respir Crit Care Med ; 164(2): 290-4, 2001 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-11463603

RESUMO

Reperfusion of the lung after hemorrhage generates free radicals such as superoxide (O(2)(.)) that may injure the lung; however, the relative importance of intracellular versus extracellular free radicals is unclear. The superoxide dismutases (SOD) are the primary enzymatic method to reduce superoxide. We examined whether lung-specific overexpression of extracellular superoxide dismutase (EC-SOD) would attenuate hemorrhage-induced lung injury. Wild-type mice and mice overexpressing the human EC-SOD gene with a lung-specific promoter were hemorrhaged by removing 30% of blood volume. After hemorrhage, the lung wet to dry weight ratios increased from 5.4 +/- 0.11 in unmanipulated control mice to 6.3 +/- 0.16 in wild-type mice, but to only 5.60 +/- 0.17 in the EC-SOD transgenic mice (p < 0.05 compared with hemorrhaged wild-type). Hemorrhage-induced lipid peroxidation, as assessed by lung F(2) isoprostanes, was lower in the EC-SOD transgenic mice (3.4 +/- 0.3 microg/lung) compared with wild-type mice (1.9 +/- 0.2 microg/lung; p < 0.05). Compared with wild-type, EC-SOD transgenic mice had attenuated the hemorrhage-induced increase in both pulmonary nuclear factor kappa B (NK-kappaB) activation (relative absorbance 1.1 +/- 0.2 for EC-SOD transgenic versus 2.5 +/- 0.1 for wild-type; p < 0.05) and myeloperoxidase activity (5.1 +/- 0.87 units/g for EC-SOD transgenic versus 11.3 +/- 1.8 units/g for wild-type; p < 0.01). Thus, overexpression of pulmonary EC-SOD in the mouse lung attenuates lung injury after hemorrhage.


Assuntos
Sequestradores de Radicais Livres/uso terapêutico , Hemorragia/complicações , Pneumopatias/tratamento farmacológico , Superóxido Dismutase/uso terapêutico , Animais , Pneumopatias/etiologia , Camundongos
12.
Proc Natl Acad Sci U S A ; 98(9): 4926-31, 2001 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-11309501

RESUMO

There is increasing evidence that sphingolipid- and cholesterol-rich microdomains (rafts) exist in the plasma membrane. Specific proteins assemble in these membrane domains and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BCtheta) of perfringolysin O (theta-toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of nontreated and Triton X-100-treated platelets after incubation with BCtheta on a sucrose gradient. BCtheta was predominantly localized in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BCtheta binds to a subpopulation of vesicles in FLDF. Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BCtheta binding to FLDF. The staining patterns of BCtheta and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BCtheta binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BCtheta binding does not cause any damage to cell membranes, indicating that BCtheta is a useful probe for the detection of membrane rafts in living cells.


Assuntos
Toxinas Bacterianas/metabolismo , Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , beta-Ciclodextrinas , Biotinilação , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Células Cultivadas , Centrifugação com Gradiente de Concentração , Ciclodextrinas/farmacologia , Endopeptidases/metabolismo , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Filipina/metabolismo , Proteínas Hemolisinas , Humanos , Microdomínios da Membrana/química , Microdomínios da Membrana/efeitos dos fármacos , Microscopia Imunoeletrônica , Sondas Moleculares/metabolismo , Octoxinol/farmacologia , Esfingomielinas/metabolismo , Especificidade por Substrato , Células Tumorais Cultivadas
13.
Mol Biol Cell ; 11(12): 4079-91, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11102509

RESUMO

Insulin treatment of fat cells results in the translocation of the insulin-responsive glucose transporter type 4, GLUT4, from intracellular compartments to the plasma membrane. However, the precise nature of these intracellular GLUT4-carrying compartments is debated. To resolve the nature of these compartments, we have performed an extensive morphological analysis of GLUT4-containing compartments, using a novel immunocytochemical technique enabling high labeling efficiency and 3-D resolution of cytoplasmic rims isolated from rat epididymal adipocytes. In basal cells, GLUT4 was localized to three morphologically distinct intracellular structures: small vesicles, tubules, and vacuoles. In response to insulin the increase of GLUT4 at the cell surface was compensated by a decrease in small vesicles, whereas the amount in tubules and vacuoles was unchanged. Under basal conditions, many small GLUT4 positive vesicles also contained IRAP (88%) and the v-SNARE, VAMP2 (57%) but not markers of sorting endosomes (EEA1), late endosomes, or lysosomes (lgp120). A largely distinct population of GLUT4 vesicles (56%) contained the cation-dependent mannose 6-phosphate receptor (CD-MPR), a marker protein that shuttles between endosomes and the trans-Golgi network (TGN). In response to insulin, GLUT4 was recruited both from VAMP2 and CD-MPR positive vesicles. However, while the concentration of GLUT4 in the remaining VAMP2-positive vesicles was unchanged, the concentration of GLUT4 in CD-MPR-positive vesicles decreased. Taken together, we provide morphological evidence indicating that, in response to insulin, GLUT4 is recruited to the plasma membrane by fusion of preexisting VAMP2-carrying vesicles as well as by sorting from the dynamic endosomal-TGN system.


Assuntos
Adipócitos/metabolismo , Endossomos/metabolismo , Insulina/farmacologia , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Rede trans-Golgi/metabolismo , Adipócitos/efeitos dos fármacos , Adipócitos/ultraestrutura , Animais , Compartimento Celular , Células Cultivadas , Endossomos/ultraestrutura , Transportador de Glucose Tipo 4 , Masculino , Microscopia Imunoeletrônica/métodos , Proteínas R-SNARE , Ratos , Vesículas Transportadoras/metabolismo , Rede trans-Golgi/ultraestrutura
14.
J Cell Sci ; 113 Pt 23: 4203-10, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11069765

RESUMO

We used an improved cryosectioning technique in combination with quantitative immunoelectron microscopy to study GLUT4 compartments in isolated rat white adipose cells. We provide clear evidence that in unstimulated cells most of the GLUT4 localizes intracellularly to tubulovesicular structures clustered near small stacks of Golgi and endosomes, or scattered throughout the cytoplasm. This localization is entirely consistent with that originally described in brown adipose tissue, strongly suggesting that the GLUT4 compartments in white and brown adipose cells are morphologically similar. Furthermore, insulin induces parallel increases (with similar magnitudes) in glucose transport activity, approximately 16-fold, and cell-surface GLUT4, approximately 12-fold. Concomitantly, insulin decreases GLUT4 equally from all intracellular locations, in agreement with the concept that the entire cellular GLUT4 pool contributes to insulin-stimulated exocytosis. In the insulin-stimulated state, GLUT4 molecules are not randomly distributed on the plasma membrane, but neither are they enriched in caveolae. Importantly, the total number of GLUT4 C-terminal epitopes detected by the immuno-gold method is not significantly different between basal and insulin-stimulated cells, thus arguing directly against a reported insulin-induced unmasking effect. These results provide strong morphological evidence (1) that GLUT4 compartments are similar in all insulin-sensitive cells and (2) for the concept that GLUT4 translocation almost fully accounts for the increase in glucose transport in response to insulin.


Assuntos
Adipócitos/metabolismo , Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , 3-O-Metilglucose/farmacocinética , Adipócitos/química , Adipócitos/ultraestrutura , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Transportador de Glucose Tipo 4 , Hipoglicemiantes/farmacologia , Imuno-Histoquímica , Insulina/farmacologia , Masculino , Microscopia Imunoeletrônica , Microtomia , Proteínas de Transporte de Monossacarídeos/análise , Ratos , Ratos Sprague-Dawley
15.
Cell ; 98(1): 81-90, 1999 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-10412983

RESUMO

We have determined the concentrations of the secretory proteins amylase and chymotrypsinogen and the membrane proteins KDELr and rBet1 in COPII- and COPI-coated pre-Golgi compartments of pancreatic cells by quantitative immunoelectron microscopy. COPII was confined to ER membrane buds and adjacent vesicles. COPI occurred on vesicular tubular clusters (VTCs), Golgi cisternae, the trans-Golgi network, and immature secretory granules. Both secretory proteins exhibited a first, significant concentration step in noncoated segments of VTC tubules and were excluded from COPI-coated tips. By contrast, KDELr and rBet1 showed a first, significant concentration in COPII-coated ER buds and vesicles and were prominently present in COPI-coated tips of VTC tubules. These data suggest an important role of VTCs in soluble cargo concentration by exclusion from COPI-coated domains.


Assuntos
Amilases/metabolismo , Quimotripsinogênio/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Pâncreas/fisiologia , Receptores de Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Animais , Proteínas de Transporte/metabolismo , Invaginações Revestidas da Membrana Celular/ultraestrutura , Proteína Coatomer , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Masculino , Microscopia Imunoeletrônica , Pâncreas/ultraestrutura , Fosfoproteínas/metabolismo , Proteínas Qc-SNARE , Ratos , Ratos Wistar
16.
Cell Biochem Biophys ; 30(1): 89-113, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10099824

RESUMO

In recent years, there have been major advances in the understanding of both the cell biology of vesicle trafficking between intracellular compartments and the molecular targeting signals intrinsic to the trafficking proteins themselves. One system to which these advances have been profitably applied is the regulation of the trafficking of a glucose transporter, GLUT4, from intracellular compartment(s) to the cell surface in response to insulin. The unique nature of the trafficking of GLUT4 and its expression in highly differentiated cells makes this a question of considerable interest to cell biologists. Unraveling the tangled web of molecular events coordinating GLUT4 trafficking will eventually lead to a greater understanding of mammalian glucose metabolism, as well as fundamental cell biological principles related to organelle biogenesis and protein trafficking.


Assuntos
Insulina/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Células 3T3 , Tecido Adiposo Marrom/metabolismo , Animais , Células Cultivadas , Endocitose , Exocitose , Transportador de Glucose Tipo 4 , Imuno-Histoquímica , Cinética , Camundongos , Modelos Biológicos , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Receptores da Transferrina/metabolismo , Transdução de Sinais
17.
J Biol Chem ; 273(40): 25880-8, 1998 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-9748263

RESUMO

UDP-galactose:ceramide galactosyltransferase (CGalT) transfers UDP-galactose to ceramide to form the glycosphingolipid galactosylceramide. Galactosylceramide is the major constituent of myelin and is also highly enriched in many epithelial cells, where it is thought to play an important role in lipid and protein sorting. Although the biochemical pathways of glycosphingolipid biosynthesis are relatively well understood, the localization of the enzymes involved in these processes has remained controversial. We here have raised antibodies against CGalT and shown by immunocytochemistry on ultrathin cryosections that the enzyme is localized to the endoplasmic reticulum and nuclear envelope but not to the Golgi apparatus or the plasma membrane. In pulse-chase experiments, we have observed that newly synthesized CGalT remains sensitive to endoglycosidase H, confirming the results of the morphological localization experiments. In protease protection assays, we show that the largest part of the protein, including the amino terminus, is oriented toward the lumen of the endoplasmic reticulum. CGalT enzyme activity required import of UDP-galactose into the lumen of the endoplasmic reticulum by a UDP-galactose translocator that is present in the Golgi apparatus of CHO cells but absent in CHOlec8 cells. Finally, we show that CGalT activity previously observed in Golgi membrane fractions in vitro, in the absence of UDP-glucose, is caused by UDP-glucose:ceramide glucosyltransferase. Therefore all galactosylceramide synthesis occurs by CGalT in vivo in the lumen of the endoplasmic reticulum.


Assuntos
Retículo Endoplasmático/enzimologia , Galactosiltransferases/química , Proteínas de Membrana/química , Animais , Transporte Biológico/fisiologia , Células CHO , Ceramidas/metabolismo , Cricetinae , Endopeptidases/farmacologia , Imunofluorescência , Galactosilceramidas/biossíntese , Gangliosídeo Galactosiltransferase , Glucosiltransferases/metabolismo , Complexo de Golgi/fisiologia , Imuno-Histoquímica , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Proteínas de Transporte de Monossacarídeos/fisiologia , Membrana Nuclear/enzimologia , Proteínas Recombinantes de Fusão/genética , Uridina Difosfato Galactose/metabolismo
18.
Am J Physiol ; 275(1): L172-83, 1998 07.
Artigo em Inglês | MEDLINE | ID: mdl-9688949

RESUMO

Monoclonal antibodies against the limiting membrane of alveolar type II cell lamellar bodies were obtained after immunization of mice with a membrane fraction prepared from lamellar bodies isolated from rat lungs. The specificity of the antibodies was investigated with Western blot analysis, indirect immunofluorescence, and electron-microscopic immunogold studies of freshly isolated or cultured alveolar type II cells, alveolar macrophages, and rat lung tissue. One of the monoclonal antibodies identified, MAb 3C9, recognized a 180-kDa lamellar body membrane (lbm180) protein. Immunogold labeling of rat lung tissue with MAb 3C9 demonstrated that lbm180 protein is primarily localized at the lamellar body limiting membrane and is not found in the lamellar body contents. Most multivesicular bodies of type II cells were also labeled, as were some small cytoplasmic vesicles. Golgi complex labeling and plasma membrane labeling were weak. The appearance of lbm180 protein by immunofluorescence in fetal rat lung cryosections correlated with the biogenesis of lamellar bodies. The lbm180 protein decreased with time in type II cells cultured on plastic. The lbm180 protein is an integral membrane protein of lamellar bodies and was also found in the pancreas and the pancreatic betaHC9 cell line but not in the rat brain, liver, kidney, stomach, or intestine. The present study provides evidence that the lbm180 protein is a lung lamellar body and/or multivesicular body membrane protein and that its antibody, MAb 3C9, will be a valuable reagent in further investigations of the biogenesis and trafficking of type II cell organelles.


Assuntos
Anticorpos Monoclonais , Membranas Intracelulares/imunologia , Membranas Intracelulares/ultraestrutura , Pulmão/imunologia , Proteínas de Membrana/análise , Organelas/imunologia , Organelas/ultraestrutura , Alvéolos Pulmonares/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Especificidade de Anticorpos , Fracionamento Celular , Células Cultivadas , Centrifugação com Gradiente de Concentração , Técnica Indireta de Fluorescência para Anticorpo , Complexo de Golgi/imunologia , Complexo de Golgi/ultraestrutura , Pulmão/citologia , Pulmão/ultraestrutura , Proteínas de Membrana/imunologia , Camundongos , Microscopia Imunoeletrônica , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/ultraestrutura , Ratos
19.
J Biol Chem ; 273(17): 10726-32, 1998 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-9553137

RESUMO

We recently cloned IRS-4, a new member of the insulin receptor substrate (IRS) family. In this study we have characterized IRS-4 in human embryonic kidney 293 cells, where it was originally discovered. IRS-4 was the predominant insulin-elicited phosphotyrosine protein in these cells. Subcellular fractionation revealed that about 50% of IRS-4 was located in cellular membranes, and immunofluorescence indicated that IRS-4 was concentrated at the plasma membrane. Immunoelectron microscopy conclusively established that a large portion of the IRS-4 was located at the cytoplasmic surface of the plasma membrane in both the unstimulated and insulin-treated states. IRS-4 was found to be associated with two src homology 2 (SH2) domain-containing proteins, phosphatidylinositol 3-kinase and Grb2, the adaptor to the guanine nucleotide exchange factor for Ras. On the other hand, no significant association was detected with two other SH2 domain proteins, the SH2-containing protein tyrosine phosphatase 2 and phospholipase Cgamma. Insulin-like growth factor I acting through its receptor was as effective as insulin in eliciting tyrosine phosphorylation of IRS-4, but interleukin 4 and epidermal growth factor were ineffective.


Assuntos
Rim/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular , Substâncias de Crescimento/metabolismo , Humanos , Proteínas Substratos do Receptor de Insulina , Rim/citologia , Rim/embriologia , Microscopia Imunoeletrônica , Fosfoproteínas/genética , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Tirosina/metabolismo , Domínios de Homologia de src
20.
Am J Respir Cell Mol Biol ; 18(4): 538-47, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9533942

RESUMO

To investigate the role of manganese-containing superoxide dismutase (MnSOD) in lung antioxidant defense, lines of transgenic B6C3 hybrid mice carrying human MnSOD transgenes under the transcriptional control of a human beta-actin promoter were established. Expression studies demonstrated that the human MnSOD transgene in line TgHMS66 is expressed and functional. The cellular distribution of the transgene product in the lungs was further examined by immunocytochemical analysis. Increased immunoreactive MnSOD was found in mitochondria of lung type I epithelial cells, type II epithelial cells, capillary endothelial cells, and fibroblasts. Furthermore, the magnitude of increase in mitochondrial labeling density of type II cells of nontransgenic, hemizygous, and homozygous transgenic littermates was proportional to the increased lung activity of MnSOD found in these mice. Transgenic mice over-expressing MnSOD did not have enhanced survival relative to controls when exposed to > 99% oxygen. However, when exposed to 90% oxygen, the transgenic mice had a small but statistically significant increase in survival time. Our results indicate that when the beta-actin promoter is used to increase activity of MnSOD it provides modest protection to B6C3 mice against hyperoxic lung injury.


Assuntos
Antioxidantes/metabolismo , Pulmão/efeitos dos fármacos , Animais , Feminino , Expressão Gênica/genética , Expressão Gênica/fisiologia , Humanos , Hiperóxia/fisiopatologia , Imuno-Histoquímica , Pulmão/química , Pulmão/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , Modelos Biológicos , Oxidantes/farmacologia , Oxigênio/farmacologia , Superóxido Dismutase/efeitos dos fármacos , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Análise de Sobrevida , Transgenes/genética
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