RESUMO
STUDY DESIGN: This is a double-blind, randomized, placebo-controlled cross-over study of melatonin in complete tetraplegia. OBJECTIVES: Tetraplegic patients have an increased risk of venous thrombosis despite prophylaxis, blunted variations in melatonin and altered circadian variation of several hemostatic markers. To examine whether melatonin could modify the regulation of hemostasis, we measured plasma melatonin and several markers of hemostasis in tetraplegic subjects with or without melatonin supplement. SETTING: The study was conducted in the Section for Spinal Cord Injury, Sunnaas Hospital, Nesoddtangen, Norway. METHODS: Six subjects with long-standing complete tetraplegia were included in this cross-over study with 2 mg of melatonin or placebo given 4 days before sampling. We also included six able-bodied men without any intervention. Plasma samples were then collected frequently during a 24-h awake/sleep cycle. The plasma concentrations of melatonin and the various markers were analyzed using linear mixed models. RESULTS: The 24-h profiles of prothrombin fragment 1+2 and von Willebrand factor, but not D-dimer, activated FVII, tissue factor pathway inhibitor and plasminogen activator inhibitor type 1, differed (P<0.05) between tetraplegic patients and able-bodied subjects. The absolute plasma concentration of activated FVII was higher (P<0.05) among the able-bodied compared with the tetraplegic groups. Supplementation of melatonin had no impact on these findings. CONCLUSIONS: We found differences in circadian variation of several hemostatic markers between able-bodied and tetraplegics. These differences were apparently unrelated to fluctuations in the melatonin concentrations, suggesting little or no role of melatonin in the regulation of hemostasis in tetraplegia. SPONSORSHIP: Financial support was provided from the Throne Holst Foundation.
Assuntos
Fármacos do Sistema Nervoso Central/uso terapêutico , Melatonina/uso terapêutico , Quadriplegia/sangue , Quadriplegia/tratamento farmacológico , Adulto , Fármacos do Sistema Nervoso Central/sangue , Medula Cervical/lesões , Ritmo Circadiano/fisiologia , Estudos Cross-Over , Método Duplo-Cego , Humanos , Masculino , Melatonina/sangue , Pessoa de Meia-Idade , Noruega , Quadriplegia/etiologia , Traumatismos da Medula Espinal/sangue , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/tratamento farmacológicoRESUMO
The plant toxin ricin is transported to the Golgi and the endoplasmic reticulum before translocation to the cytosol where it inhibits protein synthesis. The toxin can therefore be used to investigate pathways leading to the Golgi apparatus. Except for the Rab9-mediated transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network (TGN), transport routes between endosomes and the Golgi apparatus are still poorly characterized. To investigate endosome to Golgi transport, we have used here a modified ricin molecule containing a tyrosine sulfation site and quantified incorporation of radioactive sulfate, a TGN modification. A tetracycline-inducible mutant Rab9S21N HeLa cell line was constructed and characterized to study whether Rab9 was involved in transport of ricin to the TGN and, if not, to further investigate the route used by ricin. Induced expression of Rab9S21N inhibited Golgi transport of mannose 6-phosphate receptors but did not affect the sulfation of ricin, suggesting that ricin is transported to the TGN via a Rab9-independent pathway. Moreover, because Rab11 is present in the endosomal recycling compartment and the TGN, studies of transient transfections with mutant Rab11 were performed. The results indicated that routing of ricin from endosomes to the TGN occurs by a Rab11-independent pathway. Finally, because clathrin has been implicated in early endosome to TGN transport, ricin transport was investigated in cells with inducible expression of antisense to clathrin heavy chain. Importantly, endosome to TGN transport (sulfation of endocytosed ricin) was unchanged when clathrin function was abolished. In conclusion, ricin is transported from endosomes to the Golgi apparatus by a Rab9-, Rab11-, and clathrin-independent pathway.
Assuntos
Clatrina/metabolismo , Endossomos/metabolismo , Ricina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Rede trans-Golgi/metabolismo , Animais , Transporte Biológico , Células CHO , Clatrina/genética , Cricetinae , Endocitose/fisiologia , Expressão Gênica , Células HeLa , Humanos , Oligonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-beta-cyclodextrin (MbetaCD) to selectively extract cholesterol from the plasma membrane. MbetaCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MbetaCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MbetaCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MbetaCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.
Assuntos
Colesterol/fisiologia , Clatrina/fisiologia , Ciclodextrinas/farmacologia , Endocitose/fisiologia , Endossomos/fisiologia , Receptores da Transferrina/fisiologia , Transferrina/farmacocinética , beta-Ciclodextrinas , Animais , Linhagem Celular , Colesterol/isolamento & purificação , Clatrina/efeitos dos fármacos , Cães , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/ultraestrutura , Humanos , Rim , Cinética , Lipídeos de Membrana/fisiologia , Receptores da Transferrina/genética , Proteínas Recombinantes/metabolismo , Ricina/farmacocinética , Transfecção , Células Tumorais CultivadasRESUMO
UNLABELLED: Essentials Estrogens are known to influence the expression of microRNAs in breast cancer cells. We looked at microRNAs in estrogenic regulation of tissue factor pathway inhibitor α (TFPIα). Estrogen upregulated microRNA-27a/b and microRNA-494 through the estrogen receptor α. MicroRNA-27a/b and microRNA-494 are partly involved in estrogenic downregulation of TFPIα. SUMMARY: Background Tissue factor pathway inhibitor (TFPI) has been linked to breast cancer pathogenesis. We have recently reported TFPI mRNA levels to be downregulated by estrogens in a breast cancer cell line (MCF7) through the estrogen receptor α (ERα). Accumulating evidence also indicates that activation of ERα signaling by estrogens may modulate the expression of target genes indirectly through microRNAs (miRNAs). Objectives To examine if miRNAs are involved in the estrogenic downregulation of TFPIα. Methods Computational analysis of the TFPI 3'-untranslated region (UTR) identified potential binding sites for miR-19a/b, miR-27a/b, miR-494, and miR-24. Transient overexpression or inhibition of the respective miRNAs was achieved by transfection of miRNA mimics or inhibitors. Direct targeting of TFPI 3'-UTR by miR-27a/b and miR-494 was determined by luciferase reporter assay in HEK293T cells. Effects of 17α-ethinylestradiol (EE2) and fulvestrant on relative miR-27a/b, miR-494, and TFPI mRNA levels in MCF7 cells were determined by qRT-PCR and secreted TFPIα protein by ELISA. Transient knockdown of ERα was achieved by siRNA transfection. Results EE2 treatment lead to a significant increase in miR-19a, miR-27a/b, miR-494, and miR-24 mRNA levels in MCF7 cells through ERα. miR-27a/b and miR-494 mimics lead to reduced TFPI mRNA and protein levels. Luciferase assay showed direct targeting of miR-27a/b and miR-494 on TFPI mRNA. Impaired estrogen-mediated downregulation of TFPI mRNA was detected in anti-miR-27a/b and anti-miR-494 transfected cells. Conclusions Our results provide evidence that miR-27a/b and miR-494 regulate TFPIα expression and suggest a possible role of these miRNAs in the estrogen-mediated downregulation of TFPIα.
Assuntos
Regulação para Baixo , Estrogênios/química , Lipoproteínas/metabolismo , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Sítios de Ligação , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Fator Xa/química , Células HEK293 , Humanos , Células MCF-7 , Ligação Proteica , TransfecçãoRESUMO
UNLABELLED: ESSENTIALS: A hypoxic microenvironment is a common feature of tumors that may influence activation of coagulation. MCF-7 and SK-BR-3 breast cancer cells and breast cancer tissue samples were used. The results showed transcriptional repression of tissue factor pathway inhibitor expression in hypoxia. Hypoxia-inducible factor 1α may be a target for the therapy of cancer-related coagulation and thrombosis. BACKGROUND: Activation of coagulation is a common finding in patients with cancer, and is associated with an increased risk of venous thrombosis. As a hypoxic microenvironment is a common feature of solid tumors, we investigated the role of hypoxia in the regulation of tissue factor (TF) pathway inhibitor (TFPI) expression in breast cancer. OBJECTIVES: To explore the transcriptional regulation of TFPI by hypoxia-inducible factor (HIF)-1α in breast cancer cells and their correlation in breast cancer tissues. METHODS AND RESULTS: MCF-7 and SK-BR-3 breast cancer cells were cultured in 1% oxygen or treated with cobalt chloride (CoCl2 ) to mimic hypoxia. Time-dependent and dose-dependent downregulation of TFPI mRNA (quantitative RT-PCR) and of free TFPI protein (ELISA) were observed in hypoxia. Western blotting showed parallel increases in the levels of HIF-1α protein and TF. HIF-1α inhibitor abolished or attenuated the hypoxia-induced downregulation of TFPI. Luciferase reporter assay showed that both hypoxia and HIF-1α overexpression caused strong repression of TFPI promoter activity. Subsequent chromatin immunoprecipitation and mutagenesis analysis demonstrated a functional hypoxia response element within the TFPI promoter, located at -1065 to -1060 relative to the transcriptional start point. In breast cancer tissue samples, gene expression analyses showed a positive correlation between the mRNA expression of TFPI and that of HIF-1α. CONCLUSIONS: This study demonstrates that HIF-1α is involved in the transcriptional regulation of the TFPI gene, and suggests that a hypoxic microenvironment inside a breast tumor may induce a procoagulant state in breast cancer patients.
Assuntos
Coagulação Sanguínea , Neoplasias da Mama/metabolismo , Lipoproteínas/metabolismo , Oxigênio/metabolismo , Adulto , Idoso , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Hipóxia Celular , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lipoproteínas/genética , Células MCF-7 , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição Gênica , Transfecção , Microambiente TumoralRESUMO
The human hepatoma derived HepG2 cells were treated with transforming growth factor-beta (TGF-beta) or interleukin-6 (IL-6) +/- dexamethasone. The effects of treatment on lecithin:cholesterol acyltransferase (LCAT) catalytic activity and mRNA level as well as on the apolipoprotein A-I (apo A-I) mRNA level were determined. Both the LCAT activity in medium from treated HepG2 cells and the LCAT mRNA level were decreased by TGF-beta. There was no significant effect of IL-6 +/- dexamethasone, neither on the LCAT activity nor on LCAT mRNA levels. Treatment with dexamethasone alone resulted in a decreased LCAT activity in spite of a slight increase in LCAT mRNA level. The apo A-I mRNA level was reduced after treatment with TGF-beta and increased after treatment with IL-6 +/- dexamethasone and dexamethasone alone. To analyze if the effects on mRNA levels were caused by transcriptional or post-transcriptional mechanisms, run-on experiments on isolated nuclei from treated HepG2 cells and mRNA degradation experiments were performed. The transcription rate of the LCAT gene was not affected by TGF-beta, but was increased (50-100%) after treatment with IL-6 +/- dexamethasone and dexamethasone alone. The transcription rate of the apo A-I gene was reduced (20%) by TGF-beta and increased (30-60%) by IL-6 +/- dexamethasone and dexamethasone alone. Both dexamethasone and TGF-beta increased the rate of LCAT mRNA degradation. These results show that the reduced LCAT mRNA level after treatment with TGF-beta was caused by post-transcriptional mechanisms.
Assuntos
Interleucina-6/farmacologia , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Esterol O-Aciltransferase/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Apolipoproteína A-I/metabolismo , Linhagem Celular , Dexametasona/farmacologia , Regulação para Baixo , Expressão Gênica/efeitos dos fármacos , Humanos , Fosfatidilcolina-Esterol O-Aciltransferase/genética , RNA Mensageiro/metabolismo , Esterol O-Aciltransferase/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Rats were treated with hydrocortisone, dexamethasone or triamcinolone for 4 days. The effect of treatment on hepatic lipase and lecithin:cholesterol acyltransferase (LCAT) mRNA levels and catalytic activities was determined. Hepatic lipase mRNA was not affected by hydrocortisone, but was decreased after dexamethasone (-28%) and triamcinolone (-54%). Hepatic lipase activity followed the same pattern, it was not affected by hydrocortisone and lowered by dexamethasone (-38%) and triamcinolone (-70%). The LCAT mRNA level in the liver was also not affected by hydrocortisone, but increased upon treatment with dexamethasone (+22%) and triamcinolone (+72%). Plasma LCAT, determined with an excess exogenous substrate (designated LCAT-II), tended to decrease after hydrocortisone treatment (-11%) and was higher after dexamethasone (+21%) and triamcinolone (+22%). The plasma cholesterol esterification rate (designated LCAT-I), determined by incubation of the plasma at 37 degrees C, followed the same pattern. The activity ratio of hepatic lipase/LCAT-II decreased from 1 in the controls to 0.51 after dexamethasone and 0.25 in the triamcinolone-treated animals. The plasma HDL cholesterol concentration in the different groups changed oppositely to the hepatic lipase/LCAT activity ratio. It is concluded that HDL cholesterol is raised by synthetic glucocorticoids due, among other factors, to a lowered hepatic lipase and an increased plasma LCAT activity. The influence of glucocorticoids on these enzymes is, at least partly, explained by the effects on the hepatic mRNA contents.
Assuntos
Glucocorticoides/farmacologia , Lipase/metabolismo , Fígado/efeitos dos fármacos , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Animais , Arteriosclerose/tratamento farmacológico , HDL-Colesterol/sangue , Dexametasona/farmacologia , Glucocorticoides/uso terapêutico , Hidrocortisona/farmacologia , Fígado/enzimologia , Masculino , Fosfatidilcolina-Esterol O-Aciltransferase/sangue , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Triancinolona/farmacologiaRESUMO
BACKGROUND: Annexin A5 is a natural anticoagulant assumed to have thrombomodulary functions as it shields phospholipid layers from coagulation complexes. It was recently shown that the M2 haplotype within the annexin A5 gene (ANXA5) promoter reduces the transcriptional activity of the gene. In a previous report, the M2 haplotype was found to be associated with pregnancy-related venous thrombosis (VT). OBJECTIVES: To investigate whether the M1 or M2 haplotypes or other genetic variations in ANXA5 are associated with pregnancy-related VT. PATIENTS/METHODS: We investigated samples from 313 cases and 353 controls included in the VIP study, which is a case-control study of pregnancy-related VT. We analyzed tag single nucleotide polymorphisms (SNPs) selected from the CEU population (Utah Residents with Northern and Western European Ancestry) of HapMap and the M1 and the M2 haplotypes of the promoter. Odds ratios for VT were calculated for each haplotype with the wild type as the reference and for each tag SNP with the most common genotype as reference. RESULTS: We did not find any association between genetic variants in ANXA5 and the risk of pregnancy related VT, but some of the genetic variants were not in Hardy-Weinberg equilibrium. CONCLUSION: Neither the M1/M2 haplotypes nor the tag SNPs in ANXA5 were convincingly associated with pregnancy related VT, but other studies in this field are needed.
Assuntos
Anexina A5/genética , Polimorfismo de Nucleotídeo Único , Complicações Cardiovasculares na Gravidez/genética , Trombose Venosa/genética , Estudos de Casos e Controles , Bases de Dados Genéticas , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Haplótipos , Heterozigoto , Homozigoto , Humanos , Modelos Logísticos , Noruega , Razão de Chances , Fenótipo , Gravidez , Complicações Cardiovasculares na Gravidez/diagnóstico , Complicações Cardiovasculares na Gravidez/etnologia , Regiões Promotoras Genéticas , Fatores de Risco , Utah , Trombose Venosa/diagnóstico , Trombose Venosa/etnologia , População Branca/genéticaRESUMO
We have previously demonstrated that LCAT is downregulated by TGF-beta and that the regulation is post-transcriptional and involves an increased rate of RNA degradation. Sodium butyrate affects the expression of several liver-specific genes including some whose levels are altered during an acute-phase response. We have investigated the effect of sodium butyrate on LCAT activity and mRNA levels in HepG2 cells. Both the LCAT mRNA level and activity were reduced in a dose- and time-dependent manner. The reduction of LCAT mRNA levels was not, however, due to an increased degradation of processed mRNA. The transcriptional activity of the LCAT gene as seen in run-on experiments was not affected by sodium butyrate, whereas the total level of LCAT transcripts was reduced. Thus, LCAT activity and mRNA level in HepG2 cells are decreased by sodium butyrate treatment by a post-transcriptional mechanism, most likely involving increased degradation of pre-mRNA.
Assuntos
Butiratos/farmacologia , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fosfatidilcolina-Esterol O-Aciltransferase/antagonistas & inibidores , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Ácido Butírico , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/metabolismo , Relação Dose-Resposta a Droga , Meia-Vida , Humanos , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Three of the original Norwegian lecithin:cholesterol acyltransferase (LCAT) deficiency families have been investigated for mutations in the gene for lecithin:cholesterol acyltransferase by DNA sequencing of the exons amplified by the polymerase chain reaction. A single T----A transversion in codon 252 in exon 6 converting Met(ATG) to Lys(AAG) was observed in all homozygotes. In spite of the identical mutation, the disease phenotypes differed in severity. This was not reflected in the expression of LCAT in the heterozygotes.
Assuntos
Deficiência da Lecitina Colesterol Aciltransferase/genética , Feminino , Humanos , Masculino , Mutação , Noruega , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Reação em Cadeia da PolimeraseRESUMO
The exons of the lecithin:cholesterol acyltransferase (LCAT) gene in DNA samples from two of the original Swedish Fish Eye Disease patients have been amplified by polymerase chain reactions and sequenced by the dideoxy method. The two patients apparently were unrelated. In both patients a mutation in codon 10 of the first exon was found, altering proline10 to leucine. We note that the mutations causing Fish Eye Disease as well as those causing classical LCAT deficiency are spread over most of the translated gene. Why these various mutations in the same gene give rise to two different disease phenotypes remains unexplained.
Assuntos
Doenças da Córnea/genética , Éxons , Lipoproteínas/sangue , Mutação , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Sequência de Bases , Doenças da Córnea/enzimologia , DNA/sangue , DNA/genética , DNA/isolamento & purificação , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase , Suécia , SíndromeRESUMO
Plasma lipoprotein metabolism is influenced by several factors that may act by regulating the expression of proteins involved in lipoprotein metabolism, such as lecithin:cholesterol acyltransferase (LCAT). We determined the influence of several hormones and hypolipidemic drugs on hepatic LCAT gene expression and plasma LCAT activity. Liver LCAT mRNA levels were resistant to regulation by the hormones ethinylestradiol, L-thyroxine, hydrocortisone, or by the hypolipidemic drugs probucol, simvastatin, and nicotinic acid. In contrast, hepatic LCAT mRNA levels decreased to 67%, 64%, and 46% of the control levels after treatment with the fibric acid derivatives clofibrate, gemfibrozil, and fenofibrate, respectively. Fenofibrate lowered liver LCAT mRNA levels in a dose-dependent manner, which was paralleled by a decrease in plasma LCAT activity to 54% of the controls at a dose of 0.5% (w/w) in rat chow. The decrease in liver LCAT mRNA levels was maximal after 1 day, whereas the fall in plasma LCAT activity trailed by 2 days. Cessation of treatment with fenofibrate restored liver LCAT mRNA levels to control levels within 1 week. The transcription rate of the LCAT gene decreased by 25% in nuclei isolated from fenofibrate-treated rat liver, thereby indicating that hepatic LCAT gene expression is, at least partly, regulated at a transcriptional level. In contrast to the liver, brain and testis LCAT mRNA levels remained constant after treatment with fenofibrate, indicating that fibrates regulate LCAT gene expression in a tissue-selective manner.
Assuntos
Fenofibrato/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hipolipemiantes/farmacologia , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Análise de Variância , Animais , Encéfalo/enzimologia , Feminino , Hormônios/farmacologia , Fígado/enzimologia , Masculino , Especificidade de Órgãos , Fosfatidilcolina-Esterol O-Aciltransferase/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos , Testículo/enzimologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Human low density lipoprotein (LDL) was modified by exposure to cultured human endothelial cells. The endothelial cell modified LDL (EC-LDL) and control LDL (con LDL) labelled with 125I-tyramincellobiose (125I-TC) were injected into rats. Since 125I-TC is trapped in lysosomes the contribution of various organs to the catabolism of EC-LDL and con LDL could be studied. First, EC-LDL was cleared from plasma several times faster than con LDL. Then, the liver was found to be the major organ for catabolism of EC-LDL. Con LDL was distributed more evenly among the spleen, liver and adrenals as the main organs. In the liver the endothelial cells were most effective in degrading EC-LDL whereas con LDL was distributed approximately evenly between the Kupffer, endothelial and parenchymal cells. Thus, the liver endothelial cells seem to be a major pathway for catabolism of modified LDL.
Assuntos
Lipoproteínas LDL/metabolismo , Fígado/metabolismo , Animais , Células Cultivadas , Eletroforese , Endotélio/citologia , Endotélio/metabolismo , Lipoproteínas LDL/isolamento & purificação , Masculino , Ratos , Ratos Endogâmicos , Distribuição TecidualRESUMO
Formaldehyde-treated serum albumin (FSA) and acetylated low-density lipoprotein (Ac-LDL) are taken up in vivo and in vitro by the sinusoidal endothelial cells of the liver. It is not known whether both these ligands are removed by the scavenger receptor. We have studied the effect of increasing concentrations of unlabeled FSA, Ac-LDL, and endothelial cell-modified LDL (Ec-LDL) on the endocytosis of trace amounts of these ligands labeled with 125I. Uptake of 125I-Ac-LDL and 125I-Ec-LDL was strongly inhibited by FSA. Likewise, Ac-LDL and Ec-LDL reduced the uptake of 125I-FSA effectively. Our data indicate that these modified LDLs and FSA are bound to and internalized via the same receptor on liver endothelial cells.
Assuntos
Endocitose/efeitos dos fármacos , Formaldeído/farmacologia , Lipoproteínas LDL/metabolismo , Fígado/metabolismo , Albumina Sérica/metabolismo , Acetilação , Animais , Endotélio/citologia , Endotélio/metabolismo , Humanos , Ligantes , Masculino , Ratos , Ratos Endogâmicos , Receptores de LDL/metabolismoRESUMO
Overexpression of a GTPase deficient dynamin mutant in HeLa dynK44A cells causes a block in clathrin-dependent endocytosis. When endocytosis is inhibited, these cells incorporate higher levels of [(35)S]sulfate into both cellular and secreted macromolecules and larger amounts of proteoglycans such as syndecan and perlecan are immunoprecipitated from [(35)S]sulfate-labelled lysates. Gel filtration and ion-exchange chromatography revealed that the increased [(35)S]sulfate incorporation into proteoglycans was not due to significant differences in size or density of negative charge of glycosaminoglycan chains attached to proteoglycan core proteins. On the other hand, measurements of the syndecan-1 mRNA level and of [(3)H]leucine-labelled perlecan after immunoprecipitation supported the idea that the increased [(35)S]sulfate incorporation into proteoglycans was due to a selective increase in the synthesis of proteoglycan core proteins. Interestingly, the activity of protein kinase C was increased in cells expressing mutant dynamin and inhibition of protein kinase C with BIM reduced the differences in [(35)S]sulfate incorporation between cells with normal and impaired clathrin-dependent endocytosis. Thus, the activation of protein kinase C observed upon inhibition of clathrin-dependent endocytosis may be responsible for the increased synthesis of proteoglycans.
Assuntos
Clatrina/metabolismo , Endocitose/fisiologia , Proteoglicanas/biossíntese , Animais , Linhagem Celular , Condroitina ABC Liase , Cromatografia em Gel , Cromatografia por Troca Iônica , Clatrina/genética , Cricetinae , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinaminas , Fator 1 de Crescimento de Fibroblastos/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Leucina/metabolismo , Proteína Quinase C/metabolismo , Proteoglicanas/isolamento & purificação , Sulfatos/metabolismo , Radioisótopos de Enxofre , Transcrição Gênica , Transfecção , Transferrina/metabolismo , TrítioRESUMO
cDNAs encoding the ovine and bovine prion protein-like protein Doppel (Dpl) have been cloned. Sequencing revealed cDNAs of 2.85 and 3.31 kb from ovine and bovine testicular tissue, in accordance with observations of single transcripts of 3.2 and 3.6 kb on Northern blots. Sequence alignments showed a very high degree of identity between the sheep and cattle Dpl cDNAs, except for a 0.4-kb stretch in the bovine 3' untranslated region and the terminal 3' end of the sequences. The expression pattern of the Dpl gene (Prnd) in adult tissues from both species was compared by Northern blot and RT-PCR analyses. The Prnd gene was expressed strongly in testicular tissue, while low levels of expression were seen in other tissues. The open reading frame of the ovine and bovine sequences encodes a 178-amino acid protein with 95% sequence identity between the two species. Predicted structural features are in close agreement with previous reports for mouse, human, and rat Dpl.
Assuntos
Perfilação da Expressão Gênica , Príons/genética , Ovinos/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Bovinos , Clonagem Molecular , DNA Complementar/genética , Encefalopatia Espongiforme Bovina/genética , Proteínas Ligadas por GPI , Masculino , Dados de Sequência Molecular , Príons/química , RNA Mensageiro/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Scrapie/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Testículo/metabolismoRESUMO
The mechanism of cholera toxin (CT) internalization has been investigated using Caco-2 cells transfected with caveolin to induce formation of caveolae, HeLa cells with inducible synthesis of mutant dynamin (K44A) and BHK cells in which antisense mRNA to clathrin heavy chain can be induced. Here we show that endocytosis and the ability of CT to increase the level of cAMP were unaltered in caveolin-transfected cells grown either in a non-polarized or polarized manner. Treatment of Caco-2 cells with filipin reduced CT-uptake by less than 20%, suggesting that caveolae do not play a major role in the uptake. Extraction of cholesterol by methyl-beta-cyclodextrin, which removes caveolae and inhibits uptake from clathrin-coated pits, gave 30-40% reduction of CT-endocytosis. Also, CT-uptake in HeLa K44A cells was reduced by 50-70% after induction of mutant dynamin, which inhibits both caveolae- and clathrin-dependent endocytosis. These cells contain few caveolae, and nystatin and filipin had no effect on CT-uptake, indicating major involvement of clathrin-coated pits in CT-internalization. Similarly, in BHK cells, where clathrin-dependent endocytosis is blocked by induction of antisense clathrin heavy chain, the CT-uptake was reduced by 50% in induced cells. In conclusion, a large fraction of CT can be endocytosed by clathrin-dependent as well as by caveolae- and clathrin-independent endocytosis in different cell types.
Assuntos
Membrana Celular/metabolismo , Toxina da Cólera/metabolismo , Endocitose/fisiologia , Animais , Antibacterianos/farmacologia , Cavéolas/metabolismo , Caveolina 1 , Caveolinas/genética , Caveolinas/metabolismo , Linhagem Celular , Membrana Celular/ultraestrutura , Clatrina/genética , Clatrina/metabolismo , Cadeias Pesadas de Clatrina , Vesículas Revestidas por Clatrina/metabolismo , Citocalasina D/farmacologia , Dinaminas , Inibidores Enzimáticos/farmacologia , Filipina/farmacologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Genisteína/farmacologia , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , TransfecçãoRESUMO
To investigate the role of clathrin in coated vesicle formation, a cell line with inducible expression of clathrin heavy chain (CHC) antisense RNA was produced. After 18 h of CHC antisense RNA expression, the internalization of transferrin was inhibited by 90%. Although the amount of CHC was reduced by only 10%, the frequency of clathrin-coated pits at the cell surface increased by a factor of 3-5, and clathrin-coated structures also accumulated on a pleiomorphic, multivesicular, endosomal compartment. Remarkably, the coated pits were connected to the cell surface by long, tubular necks wrapped by dynamin rings, and the level of dynamin in the CHC antisense RNA-expressing cells was up-regulated 10-fold. In contrast, the amount of several other proteins associated with clathrin coat formation was unaffected. Thus, this study demonstrates that CHC antisense RNA causes accumulation of clathrin-coated pits with dynamin rings around the neck in intact cells not transfected with dynamin mutants, suggesting the existence of a previously uncharacterized functional interplay between clathrin and dynamin.
Assuntos
Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , RNA Antissenso/metabolismo , Animais , Western Blotting , Catepsina D/metabolismo , Linhagem Celular , Cricetinae , Endossomos/metabolismo , Microscopia Eletrônica , RNA Antissenso/genética , Transferrina/metabolismoRESUMO
Pulsed-field gel electrophoresis has been used to construct a long-range restriction map spanning more than 900 kb in the q22.1 region of human chromosome 16. The gene cluster containing the lecithin:cholesterol acyl transferase (LCAT) gene is located less than 480 kb from the anonymous DNA marker D16S124 in this map. The results suggest three putative CpG islands within 125 kb, in addition to the island previously shown to be located within the gene cluster. This implies a clustering of both genes and CpG islands in this chromosomal region.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 16 , Ligação Genética , Família Multigênica/genética , Fosfatidilcolina-Esterol O-Aciltransferase/genética , Marcadores Genéticos , HumanosRESUMO
We have here used diphtheria toxin as a tool to investigate the type of endocytosis used by a glycosylphosphatidylinositol-linked molecule, a glycosylphosphatidylinositol-linked version of the diphtheria toxin receptor that is able to mediate intoxication. The receptor is expressed in HeLa cells where clathrin-dependent endocytosis can be blocked by overexpression of mutant dynamin. Diphtheria toxin intoxicates cells by first binding to cell-surface receptors, then the toxin is endocytosed, and upon exposure to low endosomal pH, the toxin enters the cytosol where it inhibits protein synthesis. Inhibition of protein synthesis by the toxin can therefore be used to probe the entry of the glycosylphosphatidylinositol-linked receptor into an acidic compartment. Furthermore, degradation of the toxin can be used as an indicator of entry into the endosomal/lysosomal compartment. The data show that although expression of mutant dynamin inhibits intoxication mediated via the wild-type receptors, mutant dynamin does not affect intoxication or endocytosis and degradation of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor. Confocal microscopy demonstrated that diphtheria toxin is transported to vesicles containing EEA1, a marker for early endosomes. Biochemical and ultrastructural studies of the HeLa cells used reveal that they have very low levels of caveolin-1 and that they contain very few if any caveolae at the cell surface. Furthermore, the endocytic uptake of diphtheria toxin bound to the glycosylphosphatidylinositol-linked receptor was not reduced by methyl-beta-cyclodextrin or by nystatin which both disrupt caveolar structure and functions. Thus, uptake of a glycosylphosphatidylinositol-linked protein, in this case the diphtheria toxin receptor, into the endosomal/lysosomal system can occur independently of both caveolae and clathrin-coated vesicles.