RESUMO
Nine patients underwent arthrodesis of the knee using customised coupled nail (the Mayday arthrodesis nail), five after infected arthroplasty, one following failed arthrodesis, one for intractable anterior knee pain, one for Charcot instability and one after trauma. Comparison was made with 17 arthrodeses, eight undertaken using external fixation, four with dual compression plates, and five with long Kütntscher nails. Union was achieved in all patients (100%) at a mean time of ten months using the customised implant. There were no complications despite early weight-bearing. No further procedures were required. This contrasted with a rate of union of 53% and a complication rate of 76% with alternative techniques. Of this second group, 76% required a further operative procedure. We compared the Mayday arthrodesis nail with other techniques of arthrodesis of the knee. The differences in the need for further surgery and occurrence of complications were statistically significant (p < 0.001), and differences in the rate of nonunion and inpatient stay of less than three weeks were also significant (p < 0.05) using Fisher's exact test. We conclude that a customised coupled intramedullary nail can give excellent stability allowing early weight-bearing, and results in a high rate of union with minimal postoperative complications.
Assuntos
Artrodese/instrumentação , Pinos Ortopédicos , Prótese do Joelho , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Instabilidade Articular/cirurgia , Traumatismos do Joelho/cirurgia , Articulação do Joelho , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Dor/prevenção & controle , Desenho de Prótese , Estudos RetrospectivosRESUMO
Contracture of the proximal interphalangeal joint after surgery to excise Dupuytren's disease, despite release of the contributory structures within the finger, can be caused by flexor digitorum superficialis (FDS) contracture. We describe five cases where FDS contracture was released by intramuscular tenotomy in the distal forearm. Standard postoperative therapy for Dupuytren's fasciectomy was used and clinical review showed improved finger extension with no loss of strength. We suggest that intramuscular tenotomy of FDS in the forearm can be used safely where indicated after excision of the Dupuytren's disease.
Assuntos
Contratura de Dupuytren/cirurgia , Mãos/cirurgia , Complicações Pós-Operatórias/cirurgia , Tendões/cirurgia , Fasciotomia , Humanos , Ligamentos/cirurgia , Amplitude de Movimento Articular , Resultado do TratamentoRESUMO
A robust and reliable method was developed to measure resistant starch (RS), i.e., starch that enters the large intestine. In vivo conditions were reflected as much as possible while a user-friendly format was maintained. Parameters investigated included a-amylase concentration, pH of incubation, maltose inhibition of alpha-amylase, the need for amyloglucosidase inclusion, the effect of shaking and stirring on determined values, and problems in recovering and analyzing the RS-containing pellet. The RS values obtained were in good agreement with published in vivo data. An interlaboratory evaluation of the method has been completed (First Action Method 2002.02).
Assuntos
Fibras na Dieta/análise , Análise de Alimentos/métodos , Amido/análise , Concentração de Íons de Hidrogênio , Maltose/farmacologiaRESUMO
This prospective, randomized controlled trial assessed the use of staples for closure of the palmar skin following Dupuytren's surgery. Although staples were significantly quicker to insert than sutures, patients experienced significantly more pain on removal of staples. There was no difference in the cosmetic appearance of the wound in the two groups. We recommend use of staples for palmar wound closure following long procedures.
Assuntos
Contratura de Dupuytren/cirurgia , Técnicas de Sutura , Suturas , Adulto , Idoso , Análise de Variância , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do TratamentoRESUMO
Vasodilator actions of insulin are mediated by activation of endothelial nitric-oxide synthase (eNOS) and subsequent production of NO. Phosphatidylinositol 3-kinase and Akt play important roles in insulin-signaling pathways leading to production of NO in vascular endothelium. Here we dissected mechanisms whereby insulin activates eNOS by using the fluorescent dye DAF-2 to directly measure NO production in single cells. Insulin caused a rapid increase in intracellular NO in NIH-3T3(IR) cells transiently transfected with eNOS. The stimulation of NO production by lysophosphatidic acid (LPA) was abrogated by pretreatment of cells with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Remarkably, in the same cells, insulin-stimulated production of NO was unaffected. However, cells expressing the eNOS-S1179A mutant (disrupted Akt phosphorylation site) did not produce detectable NO in response to insulin, whereas the response to LPA was similar to that observed in cells expressing wild-type eNOS. Moreover, production of NO in response to insulin was blocked by coexpression of an inhibitory mutant of Akt, whereas the response to LPA was unaffected. Phosphorylation of eNOS at Ser(1179) was observed only in response to treatment with insulin, but not with LPA. Interestingly, platelet-derived growth factor treatment of cells activated Akt but not eNOS. Results from human vascular endothelial cells were qualitatively similar to those obtained in transfected NIH-3T3(IR) cells, although the magnitude of the responses was smaller. We conclude that insulin regulates eNOS activity using a Ca(2+)-independent mechanism requiring phosphorylation of eNOS by Akt. Importantly, phosphorylation-dependent mechanisms that enhance eNOS activity can operate independently from Ca(2+)-dependent mechanisms.
Assuntos
Cálcio/metabolismo , Endotélio Vascular/enzimologia , Insulina/metabolismo , Óxido Nítrico Sintase/metabolismo , Proteínas Serina-Treonina Quinases , Serina/química , Células 3T3 , Animais , Bovinos , Linhagem Celular , Relação Dose-Resposta a Droga , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Ativação Enzimática , Humanos , Immunoblotting , Lisofosfolipídeos/farmacologia , Camundongos , Óxido Nítrico/biossíntese , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Fatores de Tempo , TransfecçãoRESUMO
The role that Gbeta(5) regulator of G protein signaling (RGS) complexes play in signal transduction in brain remains unknown. The subcellular localization of Gbeta(5) and RGS7 was examined in rat PC12 pheochromocytoma cells and mouse brain. Both nuclear and cytosolic localization of Gbeta(5) and RGS7 was evident in PC12 cells by immunocytochemical staining. Subcellular fractionation of PC12 cells demonstrated Gbeta(5) immunoreactivity in the membrane, cytosolic, and nuclear fractions. Analysis by limited proteolysis confirmed the identity of Gbeta(5) in the nuclear fraction. Subcellular fractionation of mouse brain demonstrated Gbeta(5) and RGS7 but not Ggamma(2/3) immunoreactivity in the nuclear fraction. RGS7 and Gbeta(5) were tightly complexed in the brain nuclear extract as evidenced by their coimmunoprecipitation with anti-RGS7 antibodies. Chimeric protein constructs containing green fluorescent protein fused to wild-type Gbeta(5) but not green fluorescent fusion proteins with Gbeta(1) or a mutant Gbeta(5) impaired in its ability to bind to RGS7 demonstrated nuclear localization in transfected PC12 cells. These findings suggest that Gbeta(5) undergoes nuclear translocation in neurons via an RGS-dependent mechanism. The novel intracellular distribution of Gbeta(5).RGS protein complexes suggests a potential role in neurons communicating between classical heterotrimeric G protein subunits and/or their effectors at the plasma membrane and the cell nucleus.
Assuntos
Encéfalo/metabolismo , Núcleo Celular/metabolismo , Subunidades beta da Proteína de Ligação ao GTP , Proteínas de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP/biossíntese , Neurônios/metabolismo , Proteínas RGS/biossíntese , Proteínas RGS/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Citosol/metabolismo , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde , Immunoblotting , Imuno-Histoquímica , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Fator de Crescimento Neural/metabolismo , Células PC12 , Testes de Precipitina , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Frações Subcelulares/metabolismo , TransfecçãoRESUMO
Sorting nexins are a family of phox homology domain containing proteins that are homologous to yeast proteins involved in protein trafficking. We have identified a novel 342-amino acid residue sorting nexin, SNX15, and a 252-amino acid splice variant, SNX15A. Unlike many sorting nexins, a SNX15 ortholog has not been identified in yeast or Caenorhabditis elegans. By Northern blot analysis, SNX15 mRNA is widely expressed. Although predicted to be a soluble protein, both endogenous and overexpressed SNX15 are found on membranes and in the cytosol. The phox homology domain of SNX15 is required for its membrane association and for association with the platelet-derived growth factor receptor. We did not detect association of SNX15 with receptors for epidermal growth factor or insulin. However, overexpression of SNX15 led to a decrease in the processing of insulin and hepatocyte growth factor receptors to their mature subunits. Immunofluorescence studies showed that SNX15 overexpression resulted in mislocalization of furin, the endoprotease responsible for cleavage of insulin and hepatocyte growth factor receptors. Based on our data and the existing findings with yeast orthologs of other sorting nexins, we propose that overexpression of SNX15 disrupts the normal trafficking of proteins from the plasma membrane to recycling endosomes or the trans-Golgi network.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Endocitose , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Proteínas de Transporte/biossíntese , Proteínas de Transporte/metabolismo , Dados de Sequência Molecular , Filogenia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/biossíntese , Receptores Proteína Tirosina Quinases/metabolismo , Receptor de Insulina/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Nexinas de Classificação , Distribuição Tecidual , TransfecçãoRESUMO
Sorting nexin (SNX) 1 and SNX2 are mammalian orthologs of Vps5p, a yeast protein that is a subunit of a large multimeric complex, termed the retromer complex, involved in retrograde transport of proteins from endosomes to the trans-Golgi network. We report the cloning and characterization of human orthologs of three additional components of the complex: Vps26p, Vps29p, and Vps35p. The close structural similarity between the yeast and human proteins suggests a similarity in function. We used both yeast two-hybrid assays and expression in mammalian cells to define the binding interactions among these proteins. The data suggest a model in which hVps35 serves as the core of a multimeric complex by binding directly to hVps26, hVps29, and SNX1. Deletional analyses of hVps35 demonstrate that amino acid residues 1-53 and 307-796 of hVps35 bind to the coiled coil-containing domain of SNX1. In contrast, hVps26 binds to amino acid residues 1-172 of hVps35, whereas hVps29 binds to amino acid residues 307-796 of hVps35. Furthermore, hVps35, hVps29, and hVps26 have been found in membrane-associated and cytosolic compartments. Gel filtration chromatography of COS7 cell cytosol showed that both recombinant and endogenous hVps35, hVps29, and hVps26 coelute as a large complex ( approximately 220-440 kDa). In the absence of hVps35, neither hVps26 nor hVps29 is found in the large complex. These data provide the first insights into the binding interactions among subunits of a putative mammalian retromer complex.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Células COS , Clonagem Molecular , Humanos , Substâncias Macromoleculares , Modelos Biológicos , Dados de Sequência Molecular , Saccharomyces/genética , Homologia de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
The transcription factor FKHR is inhibited by phosphorylation in response to insulin and IGF-1 through Akt kinase. Here we show that FKHR phosphorylation in hepatocytes conforms to a hierarchical pattern in which phosphorylation of the Akt site at S(253), in the forkhead DNA binding domain, is a prerequisite for the phosphorylation of two additional potential Akt sites, T(24) and S(316). Using insulin receptor-deficient hepatocytes, we show that T(24) fails to be phosphorylated by IGF-1 receptors, suggesting that this residue is targeted by a kinase specifically activated by insulin receptors. Lack of T(24) phosphorylation is associated with the failure of IGF-1 to induce nuclear export of FKHR, and to inhibit expression of a reporter gene under the transcriptional control of the IGF binding protein-1 insulin response element. We propose that site-specific phosphorylation of FKHR is one of the mechanisms by which insulin and IGF-1 receptors exert different effects on gene expression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Receptor IGF Tipo 1/genética , Receptor de Insulina/genética , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Transformada , Proteínas de Ligação a DNA/genética , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica/efeitos dos fármacos , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Fígado , Camundongos , Fosforilação , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
Sorting nexin (SNX) 15 is a novel member of the SNX family of proteins. Although the functions of most SNXs have not yet been determined, several family members (e.g., SNX1, SNX2, SNX3, and SNX8) are orthologs of yeast proteins involved in protein trafficking. Overexpression of myc-tagged SNX15 in COS-7 cells altered the morphology of several endosomal compartments. In transient transfection experiments, myc-SNX15 was first seen in small punctate spots and small ring structures. Later, myc-SNX15 was found in larger rings. Finally, myc-SNX15 was observed in large, amorphous membrane-limited structures. These structures contained proteins from lysosomes, late endosomes, early endosomes, and the trans-Golgi network. However, the morphology of the endoplasmic reticulum and Golgi was not affected by overexpression of myc-SNX15. In myc-SNX15-overexpressing cells, the endocytosis of transferrin was severely inhibited and endocytosis of tac-trans-Golgi network (TGN) 38 and tac-furin was slowed. In addition, the recycling of internalized tac-TGN38 and tac-furin was also inhibited. Both the morphological and biochemical data indicate that SNX15 plays a crucial role in trafficking through the endocytic pathway. This is the first demonstration that a mammalian SNX protein is involved in protein trafficking.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Proteínas/metabolismo , Proteínas de Transporte Vesicular , Animais , Transporte Biológico Ativo , Células CHO , Células COS , Cricetinae , Endocitose , Endossomos/ultraestrutura , Expressão Gênica , Células HeLa , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Transferrina/metabolismoRESUMO
There are four known isoforms of the human leptin receptor (HLR) with different C-terminal cytoplasmic domains (designated by the number of unique C-terminal amino acids). In cells expressing HLR-5, -15, or -274, 15-25% of the leptin binding sites were located at the plasma membrane. In contrast, in cells expressing HLR-67, only 5% of the total binding sites were at the plasma membrane. Immunofluorescent microscopy showed that all four isoforms partially co-localized with calnexin and beta-COP, markers of the endoplasmic reticulum and the Golgi, respectively. All isoforms were also detected in an unidentified punctate compartment. All isoforms were internalized via clathrin-mediated endocytosis, but at different rates. After 20 min at 37 degrees C, 45% of a bound cohort of labeled ligand had been internalized by HLR-15, 30% by HLR-67, 25% by HLR-274, and 15% by HLR-5. Degradation of internalized leptin occurred in lysosomes. Overnight exposure to leptin down-regulated all isoforms, but to a variable extent. HLR-274 displayed the greatest down-regulation and also appeared to reach lysosomes more quickly than the other isoforms. The faster degradation of HLR-274 may help to terminate leptin signaling.
Assuntos
Proteínas de Transporte/metabolismo , Receptores de Superfície Celular , Animais , Sítios de Ligação , Células COS , Proteínas de Transporte/genética , Membrana Celular/metabolismo , Imunofluorescência , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores para Leptina , Transdução de SinaisRESUMO
Sorting nexin 1 (SNX1) is a protein that binds to the epidermal growth factor (EGF) receptor and is proposed to play a role in directing EGF receptors to lysosomes for degradation (R. C. Kurten, D. L. Cadena, and G. N. Gill, Science 272:1008-1010, 1996). We have obtained full-length cDNAs and deduced the amino acid sequences of three novel homologous proteins, which were denoted human sorting nexins (SNX2, SNX3, and SNX4). In addition, we identified a presumed splice variant isoform of SNX1 (SNX1A). These molecules contain a conserved domain of approximately 100 amino acids, which was termed the phox homology (PX) domain. Human SNX1 (522 amino acids), SNX1A (457 amino acids), SNX2 (519 amino acids), SNX3 (162 amino acids), and SNX4 (450 amino acids) are part of a larger family of hydrophilic molecules including proteins identified in Caenorhabditis elegans and Saccharomyces cerevisiae. Despite their hydrophilic nature, the sorting nexins are found partially associated with cellular membranes. They are widely expressed, although the tissue distribution of each sorting nexin mRNA varies. When expressed in COS7 cells, epitope-tagged sorting nexins SNX1, SNX1A, SNX2, and SNX4 coimmunoprecipitated with receptor tyrosine kinases for EGF, platelet-derived growth factor, and insulin. These sorting nexins also associated with the long isoform of the leptin receptor but not with the short and medium isoforms. Interestingly, endogenous COS7 transferrin receptors associated exclusively with SNX1 and SNX1A, while SNX3 was not found to associate with any of the receptors studied. Our demonstration of a large conserved family of sorting nexins that interact with a variety of receptor types suggests that these proteins may be involved in several stages of intracellular trafficking in mammalian cells.
Assuntos
Proteínas de Transporte/química , Receptores ErbB/metabolismo , Proteínas de Transporte Vesicular , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Clonagem Molecular , Proteínas Fúngicas/química , Proteínas de Helminto/química , Humanos , Dados de Sequência Molecular , Ligação Proteica/fisiologia , RNA Mensageiro/genética , Receptores de Superfície Celular/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Leptin is a hormone produced in adipose cells that regulates energy expenditure, food intake, and adiposity. In mice, we observed that circulating leptin levels increase 20-40-fold during pregnancy. Pregnant ob/ob females had no detectable serum leptin, demonstrating that the heterozygous conceptus was not the source of the leptin. However, leptin RNA and protein levels in maternal adipose tissue were not elevated. The circulating leptin was in a high molecular weight complex, suggesting that the rise in leptin was due to expression of a binding protein. Indeed, quantitative assays of serum leptin binding capacity revealed a 40-fold increase, coincident with the rise in serum leptin. Leptin binding activity reached a capacity of 207 +/- 15 nmol/liter of serum at day 18 of gestation, and half-maximal binding was observed with approximately 3 nM leptin. The binding protein was purified and partially sequenced, revealing sequence identity to the extracellular domain of the leptin receptor. We found that the placenta produces large amounts of the OB-Re isoform of leptin receptor mRNA, which encodes a soluble binding protein. Thus, the extreme hyperleptinemia of late pregnancy is attributable to binding of the leptin by a secreted form of the leptin receptor made by the placenta.
Assuntos
Proteínas de Transporte/metabolismo , Camundongos Obesos/sangue , Proteínas/metabolismo , Receptores de Superfície Celular , Tecido Adiposo/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Feminino , Expressão Gênica , Leptina , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Placenta/metabolismo , Gravidez , Ligação Proteica , RNA Mensageiro/genética , Receptores para Leptina , SolubilidadeRESUMO
Leptin, the peptide encoded by the obese gene, is secreted by adipose cells and plays a role in regulating food intake, energy expenditure, and adiposity. Because earlier studies suggested that insulin increases the expression of leptin, we investigated the effect of insulin on leptin secretion by adipose tissue. Epididymal fat pads were incubated in vitro in the presence or absence of insulin over a 4-h time course. Insulin increased leptin secretion by about 80% at all time points studied. After 10 min of insulin treatment, the amount of tissue-associated leptin was lower in insulin-stimulated tissue, presumably due to the increased secretion. At later times, both tissue-associated leptin and total leptin production were higher in insulin-treated tissue. In untreated, isolated adipose cells, immunostaining of leptin was detected in the endoplasmic reticulum by confocal microscopy. After insulin treatment, there were two populations of cells. In many cells, leptin staining became fainter and was restricted to a narrow band near the plasma membrane. However, in other cells the leptin-staining pattern was unchanged. Leptin did not colocalize with GLUT4, the glucose transporter isoform found primarily in insulin-responsive cells, in either basal or insulin-stimulated adipose cells. In this study, insulin increased both secretion and production of leptin by adipose tissue fragments. Interestingly, insulin appeared to stimulate the transport of leptin from the endoplasmic reticulum rather than acting on a pool of regulated secretory vesicles. (Endocrinology 138: 4463-4472, 1997)
Assuntos
Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Insulina/farmacologia , Proteínas Musculares , Biossíntese de Proteínas , Proteínas/metabolismo , Tecido Adiposo/citologia , Animais , Retículo Endoplasmático/química , Técnica Indireta de Fluorescência para Anticorpo , Transportador de Glucose Tipo 4 , Imuno-Histoquímica , Leptina , Masculino , Microscopia Confocal , Proteínas de Transporte de Monossacarídeos/análise , Testes de Precipitina , Proteínas/análise , Ratos , Ratos Endogâmicos , Fatores de TempoAssuntos
Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus/etiologia , Insulina/metabolismo , Proteínas/metabolismo , Receptores de Superfície Celular , Adipócitos/fisiologia , Animais , Proteínas de Transporte/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Antagonistas da Insulina , Proteínas Substratos do Receptor de Insulina , Resistência à Insulina , Leptina , Fígado/metabolismo , Camundongos , Camundongos Obesos , Obesidade/fisiopatologia , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Fosfoproteínas/metabolismo , Fosforilação , Proteínas/farmacologia , Receptor de Insulina/metabolismo , Receptores para Leptina , Transdução de SinaisRESUMO
Hepatocytes must transport newly synthesized apical membrane proteins from the basolateral to the apical plasma membrane. Our earlier morphological study showed that the apical proteins share a late (subapical) part of the transcytotic pathway with the well characterized polymeric immunoglobulin A receptor (Barr, V. A., and Hubbard, A. L. (1993) Gastroenterology 105, 554-571). Starting with crude microsomes from the livers of [35S]methionine-labeled rats, we sequentially immunoadsorbed first vesicles containing the endocytic asialoglycoprotein receptor and then (from the depleted supernatant) vesicles containing the polymeric IgA receptor. Biochemical characterization indicated that early basolateral and late endosomes were present in the first population but not in the second. Neither Golgi-, apical plasma membrane (PM)-, nor basolateral PM-derived vesicles were significant contaminants of either population. Both vesicle populations contained 35S-labeled receptor and 35S-labeled-dipeptidyl peptidase IV. Importantly, the elevated relative specific activity of the dipeptidyl peptidase (% of 35S-labeled/% immunoblotted) in the second population indicated that these vesicles must transport newly synthesized dipeptidyl peptidase IV. A distinct kind of vesicle was immunoadsorbed from a "carrier-vesicle fraction"; surprisingly, these vesicles contained little 35S-receptor and virtually no dipeptidyl peptidase IV. These results, together with previous kinetic data from in vivo experiments, are consistent with a computer-generated model predicting that newly synthesized dipeptidyl peptidase IV is delivered to basolateral endosomes, which also contain newly synthesized polymeric immunoglobulin A receptor. The two proteins are then transcytosed together to the subapical region.
Assuntos
Dipeptidil Peptidase 4/fisiologia , Organelas/fisiologia , Receptores Fc/fisiologia , Animais , Receptor de Asialoglicoproteína , Assialoglicoproteínas/metabolismo , Fracionamento Celular , Cicloeximida/farmacologia , Dipeptidil Peptidase 4/biossíntese , Endocitose , Imunoglobulina A/metabolismo , Técnicas de Imunoadsorção , Cinética , Masculino , Metionina/metabolismo , Microssomos Hepáticos/metabolismo , Modelos Biológicos , Organelas/ultraestrutura , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/fisiologia , Receptores Fc/biossíntese , Frações Subcelulares/fisiologia , Frações Subcelulares/ultraestrutura , Radioisótopos de Enxofre , Fatores de TempoRESUMO
BACKGROUND: Newly synthesized apical membrane proteins in hepatocytes go first to the basolateral membrane, from which they are retrieved and delivered to the apical domain. The goal of the present study was to identify the vesicular carriers of these molecules. METHODS: The common bile duct of rats was ligated for 10-72 hours, and then various plasma membrane proteins were localized using immunofluorescence and quantitative immuno-electron microscopy of fixed liver tissue. RESULTS: By immunofluorescence, we found intracellular punctate staining near the bile canalicular membrane of polymeric immunoglobulin A (IgA) receptor and several apical membrane proteins, but not basolateral proteins. This compartment was membrane bounded and pleiomorphic by immunoelectron microscopy. Colocalization at the electron microscopic level showed that the apical protein, dipeptidyl peptidase IV, was in the same structures as aminopeptidase N, polymeric IgA receptor, or intravenously injected horseradish peroxidase. This intracellular immunolabeling decreased after cycloheximide treatment (t1/2 = 2-2.5 hours) or reversal of the ligation for 1 hour. In the latter case, bile canalicular labeling increased. Furthermore, polymeric IgA receptor was delivered to the bile canaliculi. CONCLUSIONS: Bile duct ligation leads to an intracellular accumulation of vesicles carrying polymeric IgA receptor, several apical membrane proteins, and a fluid phase marker. These vesicles continue to fuse with the apical membrane, even during ligation.
Assuntos
Fígado/metabolismo , Proteínas de Membrana/metabolismo , Receptores Fc , Animais , Ductos Biliares , Transporte Biológico , Cicloeximida/farmacologia , Imunofluorescência , Peroxidase do Rábano Silvestre/metabolismo , Ligadura , Fígado/ultraestrutura , Masculino , Polímeros , Ratos , Ratos Sprague-Dawley , Receptores Imunológicos/metabolismo , Distribuição TecidualRESUMO
We have described the surface antigens of Mycobacterium kansasii as trehalose-containing lipooligosaccharides (LOS) which at the nonreducing "epitope" end bear a unique amino sugar containing diglycosyl unit, whereas the putative reducing end consists of an acylated alpha, alpha-trehalose-containing tetraglucosyl "core" [Hunter, S. W., Jardine, I., Yanagihara, D. L., & Brennan, P. J. (1985) Biochemistry 24, 2798-2805]. The presence of a new variation on this core, in Mycobacterium szulgai, is now reported, ----3)beta-D-Glcp-(1----6)alpha-D-Glcp(1----1)3,4,6-tri-O-acyl-2-O- Me-alpha-D-Glcp, representing the first example of an O-methyltrehalose unit in nature. The simplest of the LOS class of glycolipids in M. szulgai was defined as alpha-L-2-O-Me-Fucp(1----3)alpha-L-Rhap(1----3)alpha-L-Rh ap(1----3) beta-D-Glcp(1----6)alpha-D-Glcp(1----1)3,4,6-tri-O-acyl-2-O-Me-alpha-D-G lcp. Further glycosylation of this nonantigen, by an incompletely defined 6-deoxyhexosyl residue, confers specific antigenicity on the organism. Thus, these extraordinary structures, in a manner analogous to the better known lipopolysaccharides from rough variants of Enterobactericiae, are highly amphipathic and display variability not only in the immunogenic, distal region but also in the "invariant" lipophilic core. The contribution of these glycolipids to the hydrophobic barrier, the pseudo outer membrane of mycobacteria, is discussed.
Assuntos
Dissacarídeos/análise , Lipopolissacarídeos/isolamento & purificação , Mycobacterium/imunologia , Trealose/análise , Acilação , Configuração de Carboidratos , Sequência de Carboidratos , Espectrometria de Massas , Metilação , Dados de Sequência MolecularRESUMO
The transfer of a plasmid specifying tetracycline resistance between different derivatives of Staphylococcus aureus by phage mediated conjugation was enhanced 100- to 1000-fold by exposure of the culture to subinhibitory concentrations of beta-lactam agents. A variety of other antibiotics, including vancomycin and teicoplanin, had no such effect. The enhanced frequency of transfer was probably due to the formation of large bacterial aggregates.
Assuntos
Antibacterianos/farmacologia , Fatores R/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/efeitos dos fármacos , beta-LactamasRESUMO
One hundred cultures of methicillin-resistant Staphylococcus aureus (MRSA) were isolated from patients in a Regional Burns Unit between December 1984 and May 1985. These organisms produced large amounts of beta-lactamase which readily hydrolyzed flucloxacillin but they were sensitive to teicoplanin, dicloxacillin and cephalothin at 37.5 degrees C. The MRSA strains did not differ from methicillin-sensitive isolates in sensitivity to unsaturated fatty acids, survival in serum and plasma or desiccation. However, each culture of this strain was negative or only weakly-positive for bound coagulase and cell bound protein A. Few (eight out of 44) cultures contained plasmids and the resistance to four antibacterials was not transferable in mixed cultures. No attempt was made to isolate patients colonized with MRSA which were rarely isolated elsewhere in the hospital.