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-MolarRESUMO
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 PanorâmicaRESUMO
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 TratamentoRESUMO
Cytotoxic T lymphocytes (CTL) contain granules that are exocytosed during specific interaction with target cells (TC). In this process, the granule contents, including the lethal protein perforin, as well as granzymes, a family of serine esterases, are delivered to the TC. Information regarding the routing of these proteins towards the granule and their exact localization within the granule is of primary importance to resolve the mechanism of granule-mediated TC killing. In this study, the subcellular localization of perforin, granzymes, and known endosomal and lysosomal marker proteins was determined in human and murine CTL, by immunogold labeling of ultrathin cryosections followed by electron microscopy. Perforin and granzymes can be detected in rough endoplasmic reticulum, Golgi complex, trans-Golgi reticulum, and in all cytotoxic granules. Within the granules, they have a similar distribution and are localized not only in the so-called dense core but also over the region containing small internal vesicles. This finding implies that perforin and granzymes can be released in membrane-enveloped and/or -associated form into the intercellular cleft formed upon CTL-TC interaction. On the basis of the present evidence, additional release of these molecules in soluble form cannot be excluded. The lysosomal membrane glycoproteins lamp-1, lamp-2, and CD63, are abundantly present on the granule-delimiting outer membrane, which becomes incorporated into the CTL plasma membrane during lethal hit delivery. In contrast, the cation-dependent mannose 6-phosphate receptor, known to be present in endosomes and absent from lysosomes, is found only in a minority of the granules. Together with our previous findings that the granules are acidic and connected to the endocytic pathway, these observations define CTL granules as secretory lysosomes.
Assuntos
Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Serina Endopeptidases/metabolismo , Linfócitos T Citotóxicos/ultraestrutura , Animais , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Endocitose/fisiologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Granzimas , Humanos , Imuno-Histoquímica , Lisossomos/fisiologia , Lisossomos/ultraestrutura , Masculino , Glicoproteínas de Membrana/metabolismo , Microscopia Eletrônica , Perforina , Proteínas Citotóxicas Formadoras de Poros , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/metabolismoRESUMO
Insulin stimulates glucose transport in muscle and fat cells by causing the redistribution of a facilitative glucose transporter, GLUT-4, from an intracellular compartment to the cell surface. But what is this intracellular GLUT-4 compartment? It may be a specialized compartment, perhaps analogous to synaptic vesicles, or may simply be part of the endosomal system. Other constituents of this compartment might be regulators of GLUT-4 movement to the cell surface, and their identification should make it possible to find the link between the insulin signal transduction pathway and GLUT-4 translocation.
RESUMO
In recent years immunofluorescence microscopy has been increasingly used to study membrane traffic. In this article seven electron microscopists, all with considerable experience in using light microscopy, take a critical look at the immunofluorescence approach and argue that results obtained with this method are often overinterpreted.
RESUMO
Gold particles in colloidal solutions often vary considerably in size. The finest sols (diameter less than 15 nm), especially, are very heterogeneous, as is indicated by coefficients of variance (CV) of 25-35%. We have complexed staphylococcal protein A with gold particles (PA/Au) and then fractionated the preparations by glycerol or sucrose gradient centrifugation into very homogeneous subfractions. In this way, PA/Au probes of almost any size between 4.5 and 15 nm could be prepared. The variation of the gold particles in these fractions resulted in CV's between 9 and 16%. The reactivity of the PA/Au complex was not affected by the gradient procedure, as was shown by single- and double-labeling immunocytochemistry of ultrathin cryosections of rat pancreatic tissue.
Assuntos
Ouro , Técnicas Imunológicas , Proteína Estafilocócica A , Animais , Coloides , Microscopia Eletrônica , Pâncreas/ultraestrutura , RatosRESUMO
The influence of feeding on the ultrastruct of the frog exocrine pancreatic cell was studied by morphometrical procedures. Volume and surface of various cell structures were measured and expressed per unit cell volume. The average cellular size was not influenced by feeding. Though protein synthesis changes 5-to 10-fold (van Venrooij, W. J., and C. Poort. 1971. Biochim. Biophys. Acta. 247:468-470), no significant differences were observed in the amount of membrane that constitutes the rough endoplasmic reticulum (RER) and that represented the major part of total cellular membranes. The appearance of the RER changed. When fasted, most of its membrane was arranged in stacks of tightly packed, narrow cisternae. Within 4 h after feeding, these cisternae were separated and irregularly dilated, and ribosomes became ordered in typical rosettes on their surface. The total volume of the Golgi system increased twofold after feeding. The vesicular and tubular elements at the Golgi periphery did not change, but the volumes of the Golgi cisternae and the condensing vacuoles increased 2.5- and 6-fold, respectively. The increased in the amount of membrane present in these structures was only 1.6- and 3.5-fold, which reflects the more distended appearance of the cisternae and the rounded shape of the condensing vacuoles after feeding. Feeding halved the number of secretory granules per cell, and signs of exocytosis were more common than in fasted animals. These findings suggest that, in the frog pancreatic cell, fluctuations in the production of secretory proteins are not accompanied by an important breakdown and renewal of cellular membranes. This may favor a rapid and strong response of the cell to feeding.
Assuntos
Dieta , Jejum , Pâncreas/ultraestrutura , Rana esculenta/anatomia & histologia , Animais , Anuros , Núcleo Celular/ultraestrutura , Grânulos Citoplasmáticos/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestruturaRESUMO
This study describes the distribution of an intrinsic membrane protein, the asialoglycoprotein receptor (ASGP-R) in the trans-Golgi reticulum and compartment of uncoupling receptor and ligand (CURL) of rat liver cells. Using quantitative immunogold electron microscopy and membrane length measurements, we showed lateral nonhomogeneity of receptors in the membranes of trans-Golgi reticulum and CURL, in particular in the membranes of secretory vesicles (identified by their content of albumin and very low density lipoprotein particles) and of CURL vesicles (endosomes), including multivesicular bodies. The characteristic tubulovesicular morphology of both sorting organelles defines the transition of receptor-rich tubular membrane and the receptor-poor limiting membrane of the attached vesicles. There was a direct relationship between the size of the secretory and CURL vesicles and the density of ASGP-Rs in their membranes. Receptor density in the smallest vesicles was similar to that found in adjacent continuous tubules. The larger the vesicles, the less receptor was detectable in their membranes. We propose that the receptor molecules are excluded from the vesicle membranes by dynamic lateral redistribution. Nonrandom receptor distribution in the CURL vesicle membranes was present even at the multivesicular body stage. These observations strongly suggest the existence of barriers to ASGP-R diffusion at the junctions of tubules and vesicles. In addition, our observations suggest that ASGP-Rs are transported to the plasma membrane via a mechanism other than the normal secretory pathway.
Assuntos
Membranas Intracelulares/análise , Fígado/ultraestrutura , Organoides/análise , Receptores de Superfície Celular/análise , Receptores Imunológicos/análise , Albuminas/análise , Animais , Receptor de Asialoglicoproteína , Assialoglicoproteínas , Grânulos Citoplasmáticos/análise , Complexo de Golgi/análise , Complexo de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Fígado/análise , Masculino , Microscopia Eletrônica , Organoides/metabolismo , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/metabolismoRESUMO
Affinity-purified, monospecific rabbit antibodies against rat pancreatic alpha-amylase and bovine pancreatic alpha-chymotrypsinogen were used for immunoferritin observations of ultrathin frozen sections of mildly fixed exocrine pancreatic tissue from secretion-stimulated (pilocarpine) rats and from overnight-fasted rats and guinea pigs. The labeling patterns for both antibodies were qualitatively alike: Labeling occurred in (a) the cisternae of the rough endoplasmic reticulum (RER) including the perinuclear cisterna, in (b) the peripheral area between the RER and cis-Golgi face, and (c) all Golgi cisternae, condensing vacuoles, and secretory granules. Labeling of cytoplasmic matrix was negligible. Structures that appeared to correspond to rigid lamellae were unlabeled. Differences in labeling intensities indicated that concentration of the zymogens starts at the boundary of the RER and cis-side of the Golgi complex. These data support the view that the Golgi cisternae are involved in protein processing in both stimulated and unstimulated cells and that Golgi cisternae and condensing vacuoles constitute a functional unit.
Assuntos
Amilases/isolamento & purificação , Quimotripsinogênio/isolamento & purificação , Complexo de Golgi/enzimologia , Pâncreas/enzimologia , Animais , Jejum , Cobaias , Histocitoquímica , Imunoquímica , Masculino , Pâncreas/efeitos dos fármacos , Pâncreas/ultraestrutura , Pilocarpina/farmacologia , RatosRESUMO
Frog exocrine pancreatic tissue was studied in vitro under conditions which maintain the differences between tissues from fasted and fed animals. Sodium dodecyl sulfate (SDS) gel electrophoresis after labeling with [14C]amino acids showed that feeding stimulated the synthesis of secretory proteins to the same relative degree as the overall protein synthesis. The intracellular transport of secretory proteins was studied by electronmicroscopy autoradiography after pulse-labeling with [3H]leucine. It was found that the transport route is similar under both feeding conditions. After their synthesis in the rough endoplasmic reticulum (RER), the proteins move through the peripheral elements and cisternae of the Golgi system into the condensing vacuoles. The velocity of the transport increases considerably after feeding. When frogs are fasted, the release of labeled proteins from the RER takes greater than 90 min, whereas after feeding, this happens within 30 min. Comparable differences were observed for transport through the Golgi system. The apparent differences between the frog and mammalian pancreas in the regulation of synthesis, intracellular transport, and secretion of proteins are discussed.
Assuntos
Dieta , Jejum , Pâncreas/metabolismo , Proteínas/metabolismo , Rana esculenta/metabolismo , Animais , Anuros , Autorradiografia/métodos , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Microscopia Eletrônica , Biossíntese de ProteínasRESUMO
Insulin stimulates glucose transport in adipocytes via the rapid redistribution of the GLUT1 and GLUT4 glucose transporters from intracellular membrane compartments to the cell surface. Insulin sensitivity is dependent on the proper intracellular trafficking of the glucose transporters in the basal state. The bulk of insulin-sensitive transport in adipocytes appears to be due to the translocation of GLUT4, which is more efficiently sequestered inside the cell and is present in much greater abundance than GLUT1. The cell type and isoform specificity of GLUT4 intracellular targeting were investigated by examining the subcellular distribution of GLUT1 and GLUT4 in cell types that are refractory to the effect of insulin on glucose transport. Rat GLUT4 was expressed in 3T3-L1 fibroblasts and HepG2 hepatoma cells by DNA-mediated transfection. Transfected 3T3-L1 fibroblasts over-expressing human GLUT1 exhibited increased glucose transport, and laser confocal immunofluorescent imaging of GLUT1 in these cells indicated that the protein was concentrated in the plasma membrane. In contrast, 3T3-L1 fibroblasts expressing GLUT4 exhibited no increase in transport activity, and confocal imaging demonstrated that this protein was targeted almost exclusively to cytoplasmic compartments. 3T3-L1 fibroblasts expressing GLUT4 were unresponsive to insulin with respect to transport activity, and no change was observed in the subcellular distribution of the protein after insulin administration. Immunogold labeling of frozen ultrathin sections revealed that GLUT4 was concentrated in tubulo-vesicular elements of the trans-Golgi reticulum in these cells. Sucrose density gradient analysis of 3T3-L1 homogenates was consistent with the presence of GLUT1 and GLUT4 in discrete cytoplasmic compartments. Immunogold labeling of frozen thin sections of HepG2 cells indicated that endogenous GLUT1 was heavily concentrated in the plasma membrane. Sucrose density gradient analysis of homogenates of HepG2 cells expressing rat GLUT4 suggested that GLUT4 is targeted to an intracellular location in these cells. The density of the putative GLUT4-containing cytoplasmic membrane vesicles was very similar in HepG2 cells, 3T3-L1 fibroblasts, 3T3-L1 adipocytes, and rat adipocytes. These data indicate that the intracellular trafficking of GLUT4 is isoform specific. Additionally, these observations support the notion that GLUT4 is targeted to its proper intracellular locale even in cell types that do not exhibit insulin-responsive glucose transport, and suggest that the machinery that regulates the intracellular targeting of GLUT4 is distinct from the factors that regulate insulin-dependent recruitment to the cell surface.
Assuntos
Desoxiglucose/metabolismo , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/genética , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Transporte Biológico Ativo , Western Blotting , Carcinoma Hepatocelular , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Imunofluorescência , Vetores Genéticos , Humanos , Neoplasias Hepáticas , Camundongos , Microscopia Eletrônica , Proteínas de Transporte de Monossacarídeos/metabolismo , Plasmídeos , Ratos , TransfecçãoRESUMO
The distribution of CuZn superoxide dismutase (SOD) molecules in subcellular organelles in rat liver hepatocytes was studied using integrated biochemical, stereological, and quantitative immunocytochemical techniques. A known concentration of purified CuZn SOD in 10% gelatin was embedded alongside the liver tissue for the calculation of CuZn SOD concentrations in hepatocyte organelles and total CuZn SOD in the rat liver. Most of the CuZn SOD was located in the cytoplasmic matrix (73.1%) and in the nucleus (11.9%) with concentrations of 1.36 and 0.71 mg/cm3, respectively. Lysosomes contained the highest concentration (5.81 mg/cm3). Only low concentrations were measured in mitochondria (0.21 mg/cm3). Membrane-bound spaces of rough endoplasmic reticulum (ER), smooth ER, and the Golgi system did not contain significant concentrations of the enzyme. By adding up the concentrations in all subcellular compartments, a total liver content of CuZn SOD was established from the immunocytochemical measurements (0.386 +/- 0.028 mg/gm liver) that agreed closely with those obtained by biochemical assays (0.380 +/- 0.058 mg/gm liver). The average distances between two CuZn SOD molecules can be calculated from enzyme concentrations. It was determined that CuZn SOD molecules in the cytoplasmic matrix and nucleus were 34 and 42 nm apart, respectively. In peroxisomes and mitochondria, average intermolecular distance increased to approximately 60 nm and increased to 136 nm in smooth ER. CuZn SOD is a relatively abundant protein in the cytosol of hepatocytes and its distribution overlaps with major sites of O2- production. The efficiency of protection CuZn SOD can provide to cytosolic proteins from attacks by superoxide anion depends on the rate of O2- production, distribution of CuZn SOD relative to cytosolic proteins, and the relative reaction rates between O2- with both cytosolic proteins and CuZn SOD. Future studies of these substrate-enzyme relationships in vivo can lead to a greater understanding of how cells handle oxidant stress.
Assuntos
Fígado/enzimologia , Superóxido Dismutase/metabolismo , Animais , Especificidade de Anticorpos , Western Blotting , Imunofluorescência , Imuno-Histoquímica , Fígado/citologia , Fígado/imunologia , Organelas/enzimologia , Testes de Precipitina , Ratos , Superóxido Dismutase/imunologiaRESUMO
A semiquantitative method using immunocytochemistry on ultrathin cryosections and the protein A-gold technique was performed to study the effect of insulin on the cellular distribution of the glucose transporters in cultured 3T3-L1 adipocytes. In basal cells a substantial portion of the label was present in a tubulovesicular structure at the trans side of the Golgi apparatus, likely to represent the trans-Golgi reticulum, and in small vesicles present in the cytoplasm. Treatment with insulin induced a rapid translocation of transporters from the tubulovesicular structure to the plasma membrane. The transporter labeling of the plasma membrane increased three-fold and that of the tubulovesicular structure decreased by half. There was no effect of insulin on the degree of label in the small cytoplasmic vesicles. Removal of insulin from stimulated cells rapidly reversed the distribution of transporters to that seen in basal cells. This study thus provides the first morphological evidence for the occurrence of transporter translocation in insulin action and identifies for the first time the intracellular location of the responsive transporters.
Assuntos
Membrana Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Tecido Adiposo/citologia , Animais , Transporte Biológico/efeitos dos fármacos , Compartimento Celular/efeitos dos fármacos , Linhagem Celular , Imuno-Histoquímica , Membranas Intracelulares/metabolismo , Camundongos , Microscopia EletrônicaRESUMO
The major pathway for cytosolic constituents to enter lysosomes is by autophagy. We used two cytosolic proteins, CuZn superoxide dismutase (SOD) and carbonic anhydrase III (CAIII), as autophagic markers in male rat hepatocytes. We took advantage of the differential presence of the two proteins in autophagic vacuoles because of the high resistance of SOD to lysosomal degradation as compared with CAIII. This allows us to determine the sequence of autophagic vacuole formation. We have double immunogold-labeled SOD and CAIII in cryosections of fasted rat liver and calculated the ratios of SOD over CAIII labeling densities (SOD/CAIII) in autophagic vacuoles (AV), as compared with the cytoplasm. Different classes of AV were defined according to their SOD/CAIII, their morphology, and their additional immunolabeling for the lysosomal markers lgp120 and cathepsin D. Of all AV, 15% exhibited a cytosol-like SOD/CAIII, indicating that degradation had not yet begun. Most of these initial AV (AVi) showed two enveloping membranes. The formation of AVi was prevented by 3-methyladenine, a potent inhibitor of autophagy. Of all AV, 85% showed a SOD/CAIII that exceeded the cytosolic ratio. These single membrane-bound vacuoles were called degradative AV (AVd). Labeling for lysosomal markers allowed the characterization of AV that shared features with both AVi and AVd. These AVi/d had a cytosol-like SOD/CAIII and a double membrane, but showed some labeling for lysosomal markers. Probably these AVi/d represent the recipient compartment for lysosomal components. AVd were positive for cathepsin D and lgp120. We discerned two AVd subclasses. Early AVd with cytosol-like SOD labeling density while CAIII labeling density was consistently lower than in the cytosol. Their size was similar to AVi and AVi/d. Late AVd contained higher SOD concentrations and were mostly larger. Our findings suggest that AV acquire lysosomal constituents by fusion with small nonautophagic structures and that after subsequent elimination of the inner membrane of AVi, degradation starts resulting in the formation of early AVd and late AVd.
Assuntos
Autofagia , Anidrases Carbônicas/metabolismo , Citosol/metabolismo , Fígado/metabolismo , Superóxido Dismutase/metabolismo , Animais , Imuno-Histoquímica , Lisossomos/metabolismo , Masculino , Ratos , Ratos Wistar , Vacúolos/metabolismoRESUMO
We examined the distribution of the non-clathrin-coated vesicle-associated coat protein beta-COP in rat exocrine pancreatic cells by immunogold cytochemistry. Labeling for beta-COP was found in the Golgi region (48%) where it was associated with vesicles and buds of approximately 50 nm, showing a characteristic approximately 10-nm-thick coat. The other half of the label was present in the cytoplasm, not associated with visible coats or membranes, with a minor fraction present on small clusters of tubules and vesicles. Clathrin-coated vesicles were typically located at the trans-side of the Golgi complex, and showed a thicker coat of approximately 18 nm. Of the total beta-COP labeling over the Golgi region, 68% occurred on the cis-side, 6% on the cisternae, 17% on the rims of the cisternae, and only 9% on the trans-side. For clathrin these figures were 16, 2, 4, and 78%, respectively. At the cis-Golgi side beta-COP was present in transitional areas (TA), on so-called peripheral elements (PE), consisting of tubules and vesicles located between the cup-shaped transitional elements (TE) of the RER and the cis-most Golgi cisternae. Label for Sec23p was also present in TA but was located closer to the TE, while beta-COP labeled PE were located near the cis-Golgi cisternae. Upon energy depletion, Golgi associated beta-COP was almost exclusively (86%) in spherical aggregates of 200-500 nm in diameter, whereas the cis-side (6%), the cisternae (1%), the rims (4%) and trans-side (3%) of the Golgi complex, were barely labeled; 50% of the total label remained in the cytoplasm. The aggregates were predominantly located at the cis-side of the Golgi stack, next to, but distinct from the Sec23p positive TA, that were devoid of beta-COP and had only a few recognizable vesicles left. Incubation with aluminum fluoride resulted in fragmentation of the Golgi complex into large clusters of beta-COP positive vesicles, while 50% of the label remained in the cytoplasm, as in control cells. After 10 min of Brefeldin A treatment 91% of beta-COP was cytoplasmic and only 7% associated with membranes of the Golgi complex. The total label for beta-COP over exocrine cells remained unchanged during the incubation with either of the drugs, indicating that the drugs induce reallocation of beta-COP. Our data suggest that beta-COP plays a role in membrane transport at the cis-side of the Golgi complex.
Assuntos
Compostos de Alumínio , Complexo de Golgi/química , Proteínas de Membrana/análise , Proteínas Associadas aos Microtúbulos/análise , Pâncreas/química , Alumínio/farmacologia , Animais , Transporte Biológico , Brefeldina A , Clatrina/análise , Proteína Coatomer , Ciclopentanos/farmacologia , Metabolismo Energético , Fluoretos/farmacologia , Masculino , Microscopia Imunoeletrônica , Pâncreas/metabolismo , Pâncreas/ultraestrutura , Ratos , Ratos WistarRESUMO
Immunogold double-labeling and ultrathin cryosections were used to compare the subcellular distribution of albumin, mannose 6-phosphate receptor (MPR), galactosyltransferase, and the lysosomal enzymes cathepsin D, beta-hexosaminidase, and alpha-glucosidase in Hep G2 cells. MPR and lysosomal enzymes were found throughout the stack of Golgi cisternae and in a trans-Golgi reticulum (TGR) of smooth-surfaced tubules with coated buds and vesicles. The trans-Golgi orientation of TGR was ascertained by the co-localization with galactosyltransferase. MPR was particularly abundant in TGR and CURL, the compartment of uncoupling receptors and ligands. Both TGR and CURL also contained lysosomal enzymes, but endogenous albumin was detected in TGR only. The coated buds on TGR tubules contained MPR, lysosomal enzymes, as well as albumin. MPR and lysosomal enzymes were also found in coated pits of the plasma membrane. CURL tubules seemed to give rise to smooth vesicles, often of the multivesicular body type. In CURL, the enzymes were found in the lumina of the smooth vesicles while MPR prevailed in the tubules. These observations suggest a role of CURL in transport of lysosomal enzymes to lysosomes. When the cells were treated with the lysosomotropic amine primaquine, binding of anti-MPR to the cells in culture was reduced by half. Immunocytochemistry showed that MPR accumulated in TGR, especially in coated buds. Since these buds contain endogenous albumin and lysosomal enzymes also, these data suggest that coated vesicles originating from TGR provide for a secretory route in Hep G2 cells and that this pathway is followed by the MPR system as well.
Assuntos
Complexo de Golgi/metabolismo , Hexosefosfatos/metabolismo , Lisossomos/metabolismo , Manosefosfatos/metabolismo , Albuminas/metabolismo , Amônia/farmacologia , Animais , Proteínas de Transporte/metabolismo , Catepsina D/metabolismo , Compartimento Celular/efeitos dos fármacos , Linhagem Celular , Endocitose , Galactosiltransferases/metabolismo , Ouro , Complexo de Golgi/efeitos dos fármacos , Hexosaminidases/metabolismo , Humanos , Técnicas Imunológicas , Membranas Intracelulares/metabolismo , Neoplasias Hepáticas Experimentais , Microscopia Eletrônica , Organoides/metabolismo , Primaquina/farmacologia , Processamento de Proteína Pós-Traducional , Receptor IGF Tipo 2 , alfa-Glucosidases/metabolismo , beta-N-Acetil-HexosaminidasesRESUMO
Antibodies specific for the insulin-regulatable glucose transporter (GLUT 4) were used to immunolocalize this protein in brown adipose tissue from basal- and insulin-treated rats. Cryosections of fixed tissue were incubated with antibodies, which were subsequently labeled with Protein A/gold and examined by EM. Antibodies against albumin and cathepsin D were also used with gold particles of different sizes to identify early and late endosomes, respectively. Under basal conditions 99% of the GLUT 4 labeling was located within the cell. Labeling was predominantly in the trans-Golgi reticulum and tubulo-vesicular structures elsewhere in the cytoplasm. In insulin-stimulated cells approximately 40% of the GLUT 4 labeling was at the cell surface, where it was randomly distributed, except for occasional clustering in coated pits. Moreover, after insulin treatment, GLUT 4 was also enriched in early endosomes. We conclude that translocation of GLUT 4 to the cell surface is the major mechanism by which insulin increases glucose transport. In addition, these results suggest that in the presence of insulin GLUT 4 recycles from the cell surface, probably via the coated pit-endosome pathway that has been characterized for cell surface receptors, and also that insulin causes the redistribution of GLUT 4 by stimulating exocytosis from GLUT 4-containing tubulo-vesicular structures, rather than by slowing endocytosis of GLUT 4.
Assuntos
Tecido Adiposo Marrom/metabolismo , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Animais , Membrana Celular/metabolismo , Endocitose , Exocitose , Membranas Intracelulares/metabolismo , Masculino , Microscopia Eletrônica , Proteínas de Transporte de Monossacarídeos/imunologia , Ratos , Ratos EndogâmicosRESUMO
We used an improved cryosectioning technique in combination with immunogold cytochemistry and morphometric analysis to study the convergence of the autophagic and endocytic pathways in isolated rat hepatocytes. The endocytic pathway was traced by continuous uptake of gold tracer for various time periods, up to 45 min, while the cells were incubated in serum-free medium to induce autophagy. Endocytic structures involved in fusion with autophagic vacuoles (AV) were categorized into multivesicular endosomes (MVE) and vesicular endosomes (VE). Three types of AV--initial (AVi), intermediate (AVi/d), and degradative (AVd)--were defined by morphological criteria and immunogold labeling characteristics of marker enzymes. The entry of tracer into AV, manifested as either tracer-containing AV profiles (AV+) or fusion profiles (FP+) between AV and tracer-positive endosomal vesicles/vacuoles, was detected as early as 10 min after endocytosis. The number of AV+ exhibited an exponential increase with time. FP+ between MVE or VE and all three types of AV were observed. Among the 112 FP+ scored, 36% involved VE. Of the AV types, AVi and AVi/d were found five to six times more likely to be involved in fusions than AVd. These fusion patterns did not significantly change during the period of endocytosis (15-45 min). We conclude that the autophagic and endocytic pathways converge in a multistage fashion starting within 10 min of endocytosis. The nascent AV is the most upstream and preferred fusion partner for endosomes.
Assuntos
Autofagia , Endocitose , Vacúolos/ultraestrutura , Animais , Anidrases Carbônicas/análise , Catepsina D/análise , Células Cultivadas , Crioultramicrotomia , Endossomos/ultraestrutura , Imuno-Histoquímica , Fígado/citologia , Masculino , Metilcelulose , Compostos Organometálicos , Ratos , Ratos Wistar , Superóxido Dismutase/análiseRESUMO
Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most alpha-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the alpha-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant alpha-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of alpha-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in alpha-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (approximately 15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets.