RESUMEN
BACKGROUND AND AIM: Mitochondrial damage is commonly involved in liver injury. We have previously shown that normal mitochondria can be coated with a carrier protein to form complexes that are specifically taken up by liver cells in culture. The aim of the current study was to determine whether mitochondrial complexes could be specifically delivered to the livers of living rats by intravenous injection. METHODS: Mitochondria were harvested from fresh mouse liver, mixed with an asialoglycoprotein-based carrier, asialoorosomucoid-polylysine (AsOR-PL), and purified to form complexes. To facilitate the release of internalized mitochondria from endosomes, an endosomolytic peptide, listeriolysin O (LLO), was coupled to AsOR to form AsOR-LLO. Mitochondria alone, mitochondrial complexes with AsOR-PL, and mitochondrial complexes plus AsOR-LLO conjugate all containing the same number of mitochondria were injected intravenously. Animals were killed, and organs were removed and analyzed by quantitative polymerase chain reaction of mouse mitochondrial DNA, electron microscopy (EM), and in situ polymerase chain reaction and hybridization followed by immunohistochemical analyses. RESULTS: Calculations revealed that approximately 27% of the total injected mitochondria was detected in the liver, while less than 2% was found in spleen, and < 1% in lungs. Immunohistochemistry showed that mouse mitochondrial DNA staining was minimal with mitochondrial complexes alone, strong periportal with mitochondrial complexes co-injected with AsOR-LLO, and absent with mitochondria alone. CONCLUSIONS: Targetable mitochondrial complexes can be delivered to rat liver, and the efficiency of that process is greatly enhanced by co-injection of a targetable endosomal release agent, AsOR-LLO.
Asunto(s)
Asialoglicoproteínas/administración & dosificación , Toxinas Bacterianas/administración & dosificación , Trasplante de Células/métodos , Proteínas de Choque Térmico/administración & dosificación , Proteínas Hemolisinas/administración & dosificación , Hígado , Mitocondrias Hepáticas/trasplante , Orosomucoide/análogos & derivados , Polilisina/administración & dosificación , Animales , Proteínas Portadoras , Endosomas , Femenino , Hepatocitos/citología , Inyecciones Intravenosas , Ratones Endogámicos , Orosomucoide/administración & dosificación , Ratas Sprague-DawleyRESUMEN
BACKGROUND/AIMS: The utility of asialo-α1-acid glycoprotein (AsAGP) for assessing the fibrotic burden is unknown. This study examined the diagnostic performance of the AsAGP level for advanced liver fibrosis or cirrhosis in patients with chronic hepatitis B (CHB) or nonalcoholic fatty liver disease (NAFLD). METHODS: From July to December 2018, 48 patients with CHB and 75 with NAFLD were recruited prospectively. Transient elastography was used as the reference standard for liver fibrosis, and the cutoff liver stiffness values were defined as 10.0 kilopascal (kPa) for ≥F3 and 12.0 kPa for F4 in CHB patients, and 9.0 kPa for ≥F3 and 11.8 kPa for F4 in NAFLD patients. RESULTS: To predict stage ≥F3 and F4 fibrosis, the areas under the receiver operating characteristic curves of the AsAGP level in patients with CHB were 0.788 (95% CI 0.647-0.930; p=0.005) and 0.825 (95% CI 0.674-0.976; p=0.004), respectively. The cutoff AsAGP levels in patients with CHB that maximized the sum of the sensitivity and specificity values were 1.31 (sensitivity 100.0%, specificity 52.6%) and 1.55 (sensitivity 75.0%, specificity 80.0%), respectively. In contrast, the AsAGP level was similar regardless of the fibrosis stage in patients with NAFLD (all p>0.05 between the stages). CONCLUSIONS: The AsAGP level showed acceptable diagnostic accuracy in predicting advanced liver fibrosis and cirrhosis in patients with CHB but not in those with NAFLD. Further studies will be needed to validate the diagnostic performance of the AsAGP level in patients with NALFD.
Asunto(s)
Asialoglicoproteínas/sangre , Hepatitis B Crónica/complicaciones , Cirrosis Hepática/diagnóstico , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Orosomucoide/análogos & derivados , Adulto , Área Bajo la Curva , Diagnóstico por Imagen de Elasticidad , Femenino , Humanos , Hígado/fisiopatología , Cirrosis Hepática/complicaciones , Cirrosis Hepática/patología , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Curva ROC , Sensibilidad y Especificidad , Índice de Severidad de la EnfermedadRESUMEN
PET and noninvasive fluorescence imaging of the sialo-N-linked glycan derivatives are described. To establish the efficient labeling protocol for N-glycans and/or glycoconjugates, new labeling probes of fluorescence and 68Ga-DOTA, as the positron emission nucleus for PET, through rapid 6π-azaelectrocyclization were designed and synthesized, (E)-ester aldehydes. The high reactivity of these probes enabled the labeling of lysine residues in peptides, proteins, and even amino groups on the cell surfaces at very low concentrations of the target molecules (~10â»8 M) within a short reaction time (~5 min) to result in "selective" and "non-destructive" labeling of the more accessible amines. The first MicroPET of glycoproteins, 68Ga-DOTA-orosomucoid and asialoorosomucoid, successfully visualized the differences in the circulatory residence of glycoproteins, in the presence or absence of sialic acids. In vivo dynamics of the new N-glycoclusters, prepared by the "self-activating" Huisgen cycloaddition reaction, could also be affected significantly by their partial structures at the non-reducing end, i.e., the presence or absence of sialic acids, and/or sialoside linkages to galactose. Azaelectrocyclization chemistry is also applicable to the engineering of the proteins and/or the cell surfaces by the oligosaccharides; lymphocytes chemically engineered by sialo-N-glycan successfully target the tumor implanted in BALB/C nude mice, detected by noninvasive fluorescence imaging.
Asunto(s)
Asialoglicoproteínas/química , Compuestos Heterocíclicos/química , Oligosacáridos/química , Compuestos Organometálicos/química , Orosomucoide/análogos & derivados , Orosomucoide/química , Ácidos Siálicos/química , Imagen de Cuerpo Entero/métodos , Animales , Asialoglicoproteínas/metabolismo , Movimiento Celular , Compuestos Heterocíclicos/metabolismo , Linfocitos/química , Linfocitos/citología , Lisina/química , Lisina/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Terapia Molecular Dirigida , Oligosacáridos/metabolismo , Compuestos Organometálicos/metabolismo , Orosomucoide/metabolismo , Tomografía de Emisión de Positrones/métodos , Ácidos Siálicos/metabolismo , Coloración y Etiquetado/métodosRESUMEN
The nrF-AGP, a 51-kDa acidic glycoprotein found in surfperch (Neoditrema ransonnetii; Perciformes, Embiotocidae), is a member of the lipocalin superfamily. nrF-AGP is the major component in ovarian cavity fluid (OCF), but not in plasma of pregnant females, which suggests its potential relevance in pregnancy. However, its production in the liver, irrespective of reproductive cycle and sex, indicates that the protein also has physiological functions other than its contribution to reproduction. In the present study, Western blot analysis indicated that this protein is widely distributed in the cutaneous and intestinal mucosa, bile, and abdominal adipose tissue of fish, as well as plasma and OCF. Immunohistochemical staining of nrF-AGP was observed in hepatocytes, adipocytes, pancreatic cells, epidermal cells, and epithelial cells of ovigerous lamellae. Transcripts were detected in adipose tissue as well as hepatocytes by reverse transcription PCR analysis. This broad distribution of nrF-AGP suggests that this protein participates in various biological processes through its ability to bind to hydrophobes. After administration of biotinylated F-AGP into the ovarian cavity, the protein was detected in the cytoplasm of the intestinal epithelial cells of the fetus within 4 h. This suggests that nrF-AGP in the ovarian cavity acts as a transporter delivering maternal resources to the fetus.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Orosomucoide/análogos & derivados , Perciformes/fisiología , Viviparidad de Animales no Mamíferos/fisiología , Grasa Abdominal/metabolismo , Animales , Bilis/metabolismo , Western Blotting , Femenino , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Orosomucoide/genética , Orosomucoide/metabolismo , Ovario/metabolismo , Piel/metabolismoRESUMEN
Antibody-therapeutic agent conjugation to be delivered specifically to tumor cells is required for many target-based therapeutic strategies. In the present study, a recombinant immunotoxin was constructed by which melittin was fused to an anti-asialoglycoprotein receptor (ASGPR) single-chain variable fragment antibody (C1), and targeting ability and cytolytic efficacy of the fusion protein were studied. Our results suggested that the recombinant 29.4 kDa protein C1M was expressed in Escherichia coli as a soluble style. Binding of C1M to the surface of hepatocellular carcinoma (HCC) cells was confirmed by both immunohistochemistry and flow cytometry assays. C1M kept the hemolytic activity of melittin and exhibited cytolytic capacity to HepG2 cells at a concentration of 1.5 µg/mL, under which erythrocytes would not be lysed. The effects were greatly inhibited by coadministration with asialoorosomucoid, a natural ligand for ASGPR. These results suggested that C1M conferred targeting and ASGPR-specific cytotoxicity to HCC cells. This work makes it possible to further investigate its antihepatoma efficacy in vivo.
Asunto(s)
Receptor de Asialoglicoproteína/inmunología , Inmunotoxinas/farmacología , Meliteno/farmacología , Anticuerpos de Cadena Única , Receptor de Asialoglicoproteína/genética , Asialoglicoproteínas/farmacología , Secuencia de Bases , Carcinoma Hepatocelular/tratamiento farmacológico , Línea Celular Tumoral , Escherichia coli/genética , Hemolíticos/farmacología , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Meliteno/genética , Datos de Secuencia Molecular , Orosomucoide/análogos & derivados , Orosomucoide/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Clearance mechanisms for recombinant activated human FVII (rFVIIa; NovoSeven), a heterogeneously glycosylated protein, have yet to be fully elucidated, but may involve the liver. The effects of the gamma-carboxy glutamic acid (Gla) domain and the sialic acid content of the protein on rFVIIa clearance were investigated following intravenous administration of rFVIIa lacking the Gla domain, des(1-44) rFVIIa and asialo-rFVIIa in pharmacokinetic (PK) studies and perfused rat livers. PK parameters for both rFVIIa and des(1-44) rFVIIa had similar biphasic clearance profiles, as well as half-lives ([t(1/2)]=80 and 88 minutes, respectively), while asialo-rFVIIa was cleared quickly (t(1/2)=21 minutes) with a linear clearance profile. Perfused liver studies with all proteins (10 nM) mirrored the trends in profiles observed in the PK study. rFVIIa and des(1-44) rFVIIa were cleared to a similar extent, 41% and 35%, respectively, after 1 h, whereas plasma-derived FVII from humans (which has a higher sialylation content than rFVIIa) was cleared to a lesser extent (21%). Asialo-rFVIIa, on the other hand, was almost totally cleared and when an excess of asialo-orosomucoid was added to the perfusate, its clearance was significantly reduced (by 34%) and also for rFVIIa, albeit to a lesser extent (by 14%). Together these data suggest that carbohydrate receptor(s) (e.g. the asialoglycoprotein receptor, ASGPR) play a role in asialo-rFVIIa and rFVIIa clearance. In vivo and liver clearance data correlated well showing similar trends and indicated that rFVIIa clearance is not affected by the Gla domain, but rather by a subpopulation of N-glycosylated structures on rFVIIa.
Asunto(s)
Asialoglicoproteínas/farmacocinética , Coagulantes/farmacocinética , Factor VIIa/farmacocinética , Hígado/metabolismo , Fragmentos de Péptidos/farmacocinética , Animales , Receptor de Asialoglicoproteína/metabolismo , Asialoglicoproteínas/administración & dosificación , Asialoglicoproteínas/sangre , Asialoglicoproteínas/farmacología , Coagulantes/administración & dosificación , Coagulantes/sangre , Factor VIIa/administración & dosificación , Glicosilación , Semivida , Humanos , Inyecciones Intravenosas , Hígado/efectos de los fármacos , Masculino , Tasa de Depuración Metabólica , Orosomucoide/análogos & derivados , Orosomucoide/farmacología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/sangre , Perfusión , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/sangre , Proteínas Recombinantes/farmacocinéticaRESUMEN
The asialoglycoprotein receptor, which is abundantly and near exclusively expressed on hepatocytes, has received much attention in the design of non-viral hepatotropic DNA delivery systems. Thus, asialoglycoproteins and hexopyranosyl ligands have been coupled to DNA-binding cationic polymers and liposomes in the assembly of complexes intended for uptake by liver parenchymal cells. The aim of the study was to construct a hepatocyte-targeted multimodular liposome-based transfecting complex, in which the biotin-streptavidin interaction provides the cohesive force between the ligand asialorosomucoid and the liposome bilayer, and to evaluate its transfection capabilities in the hepatocyte-derived human transformed cell line HepG2. Dibiotinylated asialoorosomucoid was attached to cationic liposomes constructed from 3beta[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T):dioleoylphosphatidylethanolamine:biotinylcholesterylformylhydrazide (MSB1) (48:50:2 mole ratio) through streptavidin interposition. Liposome-pGL3 DNA interactions were studied by gel band shift and ethidium displacement assays. The cytotoxicity of assemblies was evaluated in the HepG2 cell line and transfection capabilities determined by measuring the activity of the transgene luciferase. Binding assays showed that all DNA was liposome associated at a DNA (negative):liposome (positive) charge ratio of 1:1. Accommodation of a streptavidin dibiotinylated asialoorosomucoid assembly was achieved at a DNA:liposome:streptavidin dibiotinylated asialoorosomucoid ratio of 1:4:9 (weight basis). Complexes showed optimal transfection activity at this ratio, which was reduced 10-fold by the presence of the competing ligand asialofetuin. The streptavidin-biotin interaction has been applied for the first time to the assembly of hepatocyte-targeted lipoplexes that display asialoorosomucoid and that are well tolerated by a human hepatoma cell line in which transfection is demonstrably achieved by receptor mediation. Favorable size and charge ratio characteristics suggest that this system may be suitable for in vivo application.
Asunto(s)
Biotina/metabolismo , Carcinoma Hepatocelular/metabolismo , Terapia Genética/métodos , Neoplasias Hepáticas/metabolismo , Nanoestructuras/química , Transfección/métodos , Transgenes , Receptor de Asialoglicoproteína/metabolismo , Asialoglicoproteínas/efectos adversos , Asialoglicoproteínas/antagonistas & inhibidores , Asialoglicoproteínas/química , Asialoglicoproteínas/metabolismo , Biotina/efectos adversos , Biotina/análogos & derivados , Biotina/química , Biotina/uso terapéutico , Biotinilación , Carcinoma Hepatocelular/terapia , Proliferación Celular/efectos de los fármacos , Colesterol/efectos adversos , Colesterol/análogos & derivados , Colesterol/química , Fetuínas , Técnicas de Transferencia de Gen , Genes Reporteros , Terapia Genética/efectos adversos , Células Hep G2 , Humanos , Ligandos , Liposomas , Neoplasias Hepáticas/terapia , Nanoestructuras/efectos adversos , Nanoestructuras/uso terapéutico , Nanoestructuras/ultraestructura , Orosomucoide/efectos adversos , Orosomucoide/análogos & derivados , Orosomucoide/antagonistas & inhibidores , Orosomucoide/química , Orosomucoide/metabolismo , Fosfatidiletanolaminas/efectos adversos , Fosfatidiletanolaminas/química , Plásmidos/efectos adversos , Plásmidos/análisis , Plásmidos/genética , Plásmidos/metabolismo , Estreptavidina/efectos adversos , Estreptavidina/metabolismo , Estreptavidina/uso terapéutico , alfa-Fetoproteínas/metabolismoRESUMEN
Human alpha(1)-acid glycoprotein (AGP), a serum glycoprotein, is thought to have anti-inflammatory effects by a mechanism that is not well understood. In this study, we investigated the pharmacokinetics of AGP in mice using enzymatically modified AGP (AGP with the sialic acids removed, asialo-AGP, and with both sialic acids and galactose removed, agalacto-AGP). It was observed that AGP was eliminated from the circulation slowly, and was mainly taken up by the liver. The elimination of labeled AGP, asialo-AGP and agalacto-AGP from the circulation was suppressed in the presence of excess unlabeled AGP, asialo-AGP and agalacto-AGP, respectively, suggesting the receptor-mediated uptake of these AGPs. Interestingly, the uptake of AGP by the liver competed with agalacto-AGP, but not with asialo-AGP, while agalacto-AGP competed with asialo-AGP. These results suggest that agalacto-AGP binds to at least two types of receptors, namely asialoglycoprotein receptor (ASGPR) and an as yet unidentified receptor that is shared with AGP, and that AGP is directly taken up by the liver through such a receptor and not via ASGPR. These findings help improve our understanding of the clearance mechanism of AGP.
Asunto(s)
Hígado/metabolismo , Orosomucoide/farmacocinética , Animales , Receptor de Asialoglicoproteína/efectos de los fármacos , Asialoglicoproteínas/síntesis química , Asialoglicoproteínas/farmacocinética , Unión Competitiva/efectos de los fármacos , Células Cultivadas , Masculino , Ratones , Ratones Endogámicos , Orosomucoide/análogos & derivados , Orosomucoide/síntesis química , Distribución TisularRESUMEN
Horseradish peroxidase (HRP) is often used as a fluid-phase marker to characterize endocytic and transcytotic processes. Likewise, it has been applied to investigate the mechanisms of biliary secretion of fluid in rat liver hepatocytes. However, HRP contains mannose residues and thus binds to mannose receptors (MRs) on liver cells, including hepatocytes. To study the role of MR-mediated endocytosis of HRP transport in hepatocytes, we determined the influence of the oligosaccharid mannan on HRP biliary secretion in the isolated perfused rat liver. A 1-minute pulse of HRP was applied followed by marker-free perfusion. HRP appeared in bile with biphasic kinetics: a first peak at 7 minutes and a second peak at 15 minutes after labeling. Perfusion with 0.8 mg/mL HRP in the presence of a twofold excess of mannan reduced the first peak by 41% without effect on the second one. Together with recently published data on MR expression in rat hepatocytes this demonstrates two different mechanisms for HRP transcytosis: a rapid, receptor-mediated transport and a slower fluid-phase transport.
Asunto(s)
Endocitosis , Hepatocitos/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/metabolismo , Receptores de Superficie Celular/metabolismo , Reología , Animales , Asialoglicoproteínas/metabolismo , Bilis/metabolismo , Biomarcadores , Dextranos/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Cinética , Ligandos , Masculino , Mananos/metabolismo , Receptor de Manosa , Orosomucoide/análogos & derivados , Orosomucoide/metabolismo , Ratas , Transducción de SeñalRESUMEN
The asialoglycoprotein receptor (ASGPR), an endocytotic cell surface receptor expressed by hepatocytes, is triggered by triantennary binding to galactose residues of macromolecules such as asialoorosomucoid (ASOR). The capacity of this receptor to import large molecules across the cellular plasma membrane makes it an enticing target for receptor-mediated drug delivery to hepatocytes and hepatoma cells via ASGPR-mediated endocytosis. This study describes the preparation and characterization of (125)I-ASOR, and its utility in the assessment of ASGPR expression by HepG2, HepAD38 and Huh5-2 human hepatoma cell lines. ASOR was prepared from human orosomucoid, using acid hydrolysis to remove sialic acid residues, then radioiodinated using iodogen. (125)I-ASOR was purified by gel column chromatography and characterized by SDS-PAGE electrophoresis. The ASOR yield by acid hydrolysis was 75%, with approximately 87 % of the sialic acid residues removed. Electrophoresis and gel chromatography demonstrated substantial differences in (125)I-ASOR quality depending on the method of radioiodination. ASGPR densities per cell were estimated at 76,000 (HepG2), 17,000 (HepAD38) and 3,000 (Huh-5-2). (125)I-ASOR binding to ASGPR on HepG2 cells was confirmed through galactose- and EDTA- challenge studies. It is concluded that (125)I-ASOR is a facilely-prepared, stable assay reagent for ASGPR expression if appropriately prepared, and that HepG2 cells, but not HepAD38 or Huh-5-2 cells, are suitable for studies exploiting the endocytotic ASGPR.
Asunto(s)
Receptor de Asialoglicoproteína/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Antivirales/metabolismo , Asialoglicoproteínas/metabolismo , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Endocitosis , Humanos , Radioisótopos de Yodo , Orosomucoide/análogos & derivados , Orosomucoide/metabolismoRESUMEN
Following endocytosis, internalized molecules are found within intracellular vesicles and tubules that move along the cytoskeleton and undergo fission, as demonstrated here using primary cultured rat hepatocytes. Although the use of depolymerizing drugs has shown that the cytoskeleton is not required to segregate endocytic protein, many studies suggest that the cytoskeleton is involved in the segregation of protein in normal cells. To investigate whether cytoskeletal-based movement results in the segregation of protein, we tracked the contents of vesicles during in vitro microscopy assays. These studies showed that the addition of ATP causes fission of endocytic contents along microtubules, resulting in the segregation of proteins that are targeted for different cellular compartments. The plasma membrane proteins, sodium (Na+) taurocholate cotransporting polypeptide (ntcp) and transferrin receptor, segregated from asialoorosomucoid (ASOR), an endocytic ligand that is targeted for degradation. Epidermal growth factor receptor, which is degraded, and the asialoglycoprotein receptor, which remains partially bound to ASOR, segregated less efficiently from ASOR. Vesicles containing ntcp and transferrin receptor had reduced fission in the absence of ASOR, suggesting that fission is regulated to allow proteins to segregate. A single round of fission resulted in 6.5-fold purification of ntcp from ASOR, and 25% of the resulting vesicles were completely depleted of the endocytic ligand.
Asunto(s)
Citoesqueleto/metabolismo , Endocitosis/fisiología , Endosomas/metabolismo , Microtúbulos/metabolismo , Animales , Receptor de Asialoglicoproteína/metabolismo , Asialoglicoproteínas/metabolismo , Células Cultivadas , Receptores ErbB/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Nocodazol/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Orosomucoide/análogos & derivados , Orosomucoide/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Transporte de Proteínas/fisiología , Ratas , Receptores de Transferrina/metabolismo , Simportadores/metabolismo , Moduladores de Tubulina/metabolismoRESUMEN
Ribavirin is a synthetic nucleoside analog that is used for the treatment of hepatitis C virus (HCV) infection. Its primary toxicity is hemolytic anemia, which sometimes necessitates dose reduction or discontinuation of therapy. Selective delivery of ribavirin into liver cells would be desirable to enhance its antiviral activity and avoid systemic side effects. One approach to liver-specific targeting is conjugation of the ribavirin with asialoglycoprotein that is taken up specifically by liver cells. Human uridine-cytidine kinase-1 (UCK-1) was used for ribavirin phosphorylation to its monophosphate form. 1-Ethyl-3-diisopropylaminocarbodiimide (EDC) was used as a coupling agent. The best results were obtained using direct conjugation protocol with a molar ratio of 6.5 ribavirin monophosphate (RMP) molecules per one asialoorosomucoid (AsOR) molecule. Our findings show that ribavirin is a potential substrate of UCK-1, and RMP formed could be chemically coupled to AsOR to form a conjugate for liver specific targeting.
Asunto(s)
Asialoglicoproteínas/química , Sistemas de Liberación de Medicamentos , Hepatitis C/enzimología , Hígado/enzimología , Nucleósido-Fosfato Quinasa/química , Orosomucoide/análogos & derivados , Ribavirina/química , Anemia Hemolítica/inducido químicamente , Anemia Hemolítica/enzimología , Asialoglicoproteínas/uso terapéutico , Hepacivirus , Hepatitis C/complicaciones , Humanos , Hígado/virología , Nucleósido-Fosfato Quinasa/metabolismo , Orosomucoide/química , Orosomucoide/uso terapéutico , Fosforilación , Ribavirina/efectos adversos , Ribavirina/uso terapéuticoRESUMEN
OBJECTIVE: To testify that the asialoorosomucoid (ASOR) prepared by us has liver-targeting specificity and to investigate its pharmacokinetic characteristics. METHODS: The distribution of 125I-ASOR in vivo was determined by single photon emission computed tomography (SPECT) and immunohistochemical technique after 125I-ASOR was injected into Sprague-Dawley (S-D) rats through their caudal veins. In vitro, different doses of pEGFP-N1 plasmid were transfected into both HepG2 cells and HT1080 cells with the use of ASOR-poly-L-lysine. At 24 and 48 h after transfection, the expression of green fluorescent protein (GFP) was determined under fluorescent microscope. Pharmacokinetic parameters were calculated according to two-compartment open system model with first-order kinetics. RESULTS: SPECT images showed that 125I-ASOR was located only in liver/stomach and root of caudal vein/bladder at 10 min after injection. The 125I-ASOR radioactivities of organs taken out from S-D rats were different at different times, and about 63% of 125I-ASOR was located in the liver at 10 min after injection. At 30 min after injection a peak of radioactivity was seen in stomach. The times of these two radioactivity peaks were different. Immunohistochemical study of liver frozen sections showed that ASOR was combined mainly with hepatocyte membrane, especially in areas with rich blood flow. In vitro study showed that ASOR targeted specifically cells with asialoglycoprotein receptor (ASGr). GFP expression was detected in HepG2 cells but not in HT1080 cells. Furthermore, the more quantity of pEGFP-N1 transfected and the longer expression time, the higher GFP expression level was in HepG2 cells. The 125I-ASOR pharmacokinetics equation for liver was Ct=662216e-3.362t+8896e-2343t. 125I-ASOR was excreted from liver slowly after an initial rapid decrease. The pharmacokinetic equation for stomach was Ct=-114815e-1.7t+1148153e-15t and the half-life of 125I-ASOR in stomach was 4.62 h. CONCLUSIONS: ASOR prepared by us could be an efficient gene transfer vector, ASOR was distributed mainly in the liver and stomach and had high targeting specificity to hepatocytes or hepatic originating cells.
Asunto(s)
Receptor de Asialoglicoproteína/efectos de los fármacos , Asialoglicoproteínas/farmacocinética , Glicoproteínas/farmacocinética , Hígado/metabolismo , Orosomucoide/análogos & derivados , Estómago/efectos de los fármacos , Animales , Asialoglicoproteínas/química , Técnicas de Transferencia de Gen , Vectores Genéticos , Glicoproteínas/química , Hepatocitos/metabolismo , Inyecciones Intravenosas , Radioisótopos de Yodo , Orosomucoide/química , Orosomucoide/farmacocinética , Ratas , Ratas Sprague-Dawley , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
To isolate mutant liver cells defective in the endocytic pathway, a selection strategy using toxic ligands for two distinct membrane receptors was utilized. Rare survivors termed trafficking mutants (Trf2-Trf7) were stable and more resistant than the parental HuH-7 cells to both toxin conjugates. They differed from the previously isolated Trf1 HuH-7 mutant as they expressed casein kinase 2 alpha'' (CK2alpha'') which is missing from Trf1 cells and which corrects the Trf1 trafficking phenotype. Binding of (125)I-asialoorosomucoid (ASOR) and cell surface expression of asialoglycoprotein receptor (ASGPR) were reduced approximately 20%-60% in Trf2-Trf7 cells compared to parental HuH-7, without a reduction in total cellular ASGPR. Based on (125)I-transferrin binding, cell surface transferrin receptor activity was reduced between 13% and 88% in the various mutant cell lines. Distinctive phenotypic traits were identified in the differential resistance of Trf2-Trf7 to a panel of lectins and toxins and to UV light-induced cell death. By following the endocytic uptake and trafficking of Alexa(488)-ASOR, significant differences in endosomal fusion between parental HuH-7 and the Trf mutants became apparent. Unlike parental HuH-7 cells in which the fusion of endosomes into larger vesicles was evident as early as 20 min, ASOR endocytosed into the Trf mutants remained within small vesicles for up to 60 min. Identifying the biochemical and genetic mechanisms underlying these phenotypes should uncover novel and unpredicted protein-protein or protein-lipid interactions that orchestrate specific steps in membrane protein trafficking.
Asunto(s)
Endocitosis/genética , Hepatocitos/citología , Hígado/citología , Apoptosis/efectos de la radiación , Receptor de Asialoglicoproteína/metabolismo , Asialoglicoproteínas/metabolismo , Toxinas Bacterianas/farmacología , Quinasa de la Caseína II/fisiología , Separación Celular/métodos , Toxina Diftérica/farmacología , Endocitosis/efectos de la radiación , Humanos , Orosomucoide/análogos & derivados , Orosomucoide/metabolismo , Transporte de Proteínas/genética , Pseudomonas/química , Receptores de Transferrina/metabolismo , Ricina/farmacología , Rayos Ultravioleta , Aglutininas del Germen de Trigo/farmacologíaRESUMEN
Early endocytic vesicles loaded with Texas Red asialoorosomucoid were prepared from mouse liver. These vesicles bound to microtubules in vitro, and upon ATP addition, they moved bidirectionally, frequently undergoing fission into two daughter vesicles. There was no effect of vanadate (inhibitor of dynein) on motility, whereas 5'-adenylylimido-diphosphate (kinesin inhibitor) was highly inhibitory. Studies with specific antibodies confirmed that dynein was not associated with these vesicles and that Kif5B and the minus-end kinesin Kifc1 mediated their plus- and minus-end motility, respectively. More than 90% of vesicles associated with Kifc1 also contained Kif5B, and inhibition of Kifc1 with antibody resulted in enhancement of plus-end-directed motility. There was reduced vesicle fission when either Kifc1 or Kif5B activity was inhibited by antibody, indicating that the opposing forces resulting from activity of both motors are required for fission to occur. Immunoprecipitation of native Kif5B by FLAG antibody after expression of FLAG-Kifc1 in 293T cells indicates that these two motors can interact with each other. Whether they interact directly or through a complex of potential regulatory proteins will need to be clarified in future studies. However, the present study shows that coordinated activity of these kinesins is essential for motility and processing of early endocytic vesicles.
Asunto(s)
Endocitosis/fisiología , Cinesinas/metabolismo , Hígado/metabolismo , beta Carioferinas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos , Asialoglicoproteínas/metabolismo , Colorantes Fluorescentes , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Técnicas In Vitro , Hígado/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Motoras Moleculares/antagonistas & inhibidores , Proteínas Motoras Moleculares/deficiencia , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/metabolismo , Movimiento , Orosomucoide/análogos & derivados , Orosomucoide/metabolismo , Xantenos , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/deficiencia , beta Carioferinas/genéticaRESUMEN
Serum asialoglycoprotein (desialylated glycoproteins) concentrations have been reported to be elevated in patients with hepatic disease as compared with that of normal subjects. We recently developed a solid-phase sandwich assay for asialo alpha1-acid glycoprotein (AsAGP) as a representative of the serum asialoglycoproteins and evaluated the utility of this AsAGP as a diagnostic marker for liver cirrhosis (LC) and/or hepatocellular carcinoma (HCC). In this study, we developed a rapid, one-step immunochromatographic strip capable of specifically detecting AsAGP in serum specimens. We have produced a monoclonal antibody (mAb) to AGP, and based on ELISA and Western blot analysis, we have selected four hybridoma clones which generated mAbs to recognize AsAGP. In the immunochromatographic strip test, one mAb was used for conjugation with colloidal gold microparticles. Ricinus communis agglutinin (RCA) was immobilized onto a nitrocellulose membrane strip to form a result line in the path of chromatographic migration. Likewise, a control line was created above the result line by the immobilization of anti-mouse IgG. A serum specimen was then applied to the sample pad. The AsAGP in the sample specifically bound to the microparticles via mAb (As16.89) and co-migrated upward until the AsAGP was sandwiched with the immobilized lectin (RCA), revealing a visible result line. The colloidal gold microparticles without bound AsAGP continued to migrate, forming a visible control line. Thus, an AsAGP-positive specimen (>1.5 microg/mL) yielded a result line and a control line, whereas an AsAGP-negative specimen (<1.5 microg/mL) produced only a single control line. The entire test procedure was completed in less than 5 min. In order to examine the reliability of the testing procedures, we carried out the immunochromatographic strip test with 102 serum samples and compared the results of these tests with those obtained by ELISA. The two methods showed excellent correlation, with 83-100% above/below the cut-off value (1.5 microg/mL). Therefore, we concluded that the results of the immunochromatographic test are in excellent accordance with those of the sandwich ELISA.
Asunto(s)
Asialoglicoproteínas/sangre , Cromatografía/métodos , Inmunoensayo/métodos , Hepatopatías/sangre , Orosomucoide/análogos & derivados , Animales , Anticuerpos Monoclonales , Asialoglicoproteínas/inmunología , Biomarcadores/sangre , Carcinoma Hepatocelular/sangre , Colodión , Ensayo de Inmunoadsorción Enzimática , Oro Coloide , Humanos , Cirrosis Hepática/sangre , Hepatopatías/diagnóstico , Neoplasias Hepáticas/sangre , Ratones , Orosomucoide/inmunología , Lectinas de Plantas , ConejosRESUMEN
Receptor mediated gene delivery is a new gene transfer strategy. Asialoglycoprotein receptor (ASGP-R), the receptor of asialoorosomucoid (Asor), is specially expressed on the surface of hepatocyte. In this paper, the nuclide 131I was combined with Asor to form a kind of soluble nuclide-protein complex, which can be specifically endocytosed into hepatocyte by ASGP-R. After in travenous injection of the complex into experimental animals, the deposition of Asor in vivo and the targeting quality of hepatocyte was detected by ECT. This research testified the feasibility of targeting Asor complex delivery to hepatocyte mediated by ASGP-R in vivo, and provided foundation for the genetic diagnosis and gene therapy of hepatic cell-related diseases.
Asunto(s)
Receptor de Asialoglicoproteína/efectos de los fármacos , Asialoglicoproteínas/metabolismo , Sistemas de Liberación de Medicamentos , Hepatocitos/metabolismo , Neoplasias Hepáticas/patología , Orosomucoide/análogos & derivados , Animales , Receptor de Asialoglicoproteína/fisiología , Sistemas de Liberación de Medicamentos/métodos , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Estudios de Factibilidad , Humanos , Radioisótopos de Yodo , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Orosomucoide/metabolismo , Unión Proteica , Conejos , Células Tumorales CultivadasRESUMEN
The binding of flunitrazepam (FNZP) by human alpha1-acid glycoprotein (hAGP) and the relationships between the extent of drug binding and desialylation and the genetic variants of hAGP were examined. The photolabeling specificity of [3H]FNZP was confirmed by findings in which other hAGP-binding ligands inhibited the formation of covalent bonds between [3H]FNZP and hAGP. The photolabeling of asialo-hAGP suggested that sialic acid does not involve in the binding of [3H]FNZP. No difference in the labeling could be found between the F1*S variants and A variant. Similarly, FNZP did not show a difference in binding affinity to the two genetic variants of hAGP. Sequence analysis of the photolabeled peptide indicated a sequence corresponding to Tyr91-Arg105 of hAGP.
Asunto(s)
Sitios de Unión , Flunitrazepam/metabolismo , Orosomucoide/química , Etiquetas de Fotoafinidad/metabolismo , Secuencia de Aminoácidos , Asialoglicoproteínas/metabolismo , Bromuro de Cianógeno , Variación Genética , Humanos , Datos de Secuencia Molecular , Orosomucoide/análogos & derivados , Orosomucoide/genética , Orosomucoide/metabolismo , Fragmentos de Péptidos/química , TripsinaRESUMEN
AIM: To construct different conformations of a plasmid DNA/vector complex (pcDNA3.1/IFN-gamma-ASOR-PLL) and transfect cells of the hepatoma cell line BEL7402 to investigate the optimal conformation of the complex for improved expression efficiency in the target cell. METHODS: Double-distilled water and adjuvant were added to the naked pcDNA3.1/IFN-gamma, target vector ASOR-PLL and the ASOR-PLL-pcDNA3.1/IFN-gamma complex to create different conformations; molecules that were transfected into BEL7402 cells and the expression efficiency was determined by measuring the IFN-g concentration in the culture supernatant by ELISA. RESULTS: Naked pcDNA3.1/IFN-gamma DNA distributed linearly in double-distilled water and condensed into a mica configuration in adjuvant; ASOR-PLL had a net-like distribution without adjuvant and a spider-like form in the adjuvant-treated group; the ASOR-PLL-pcDNA3.1/IFN-g complex had a divaricate form without adjuvant, but a bead-like or granular conformation in 0.1 and 0.2 mol/L of adjuvant, a homogeneous bacilliform or chromatoid-shaped conformation in 0.3 mol/L adjuvant, and varied shapes in 0.4 and 0.5 mol/L adjuvant. The supernatant IFN-gamma expression in the bacilliform/chromatoid conformation complex group was the highest among the different conformation groups and controls. When chloroquine was added the supernatant IFN-gamma concentration increased in the liposome group and decreased in the bacilliform/chromatoid conformation group . CONCLUSIONS: The two structural molecules and their complex, ASOR-PLL-pcDNA3.1/IFN-gamma, were adjustable in the liquid mode. The specific bacilliform/chromatoid conformation of complex was lysosome enzyme-resistant and could play an active role in improving the efficiency of gene expression. The hypothesis that a chromosome-like conformation of the target gene molecule is involved in enhancing exogenous gene expression is proposed.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Asialoglicoproteínas/química , Asialoglicoproteínas/farmacología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Interferón gamma/química , Interferón gamma/farmacología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Orosomucoide/análogos & derivados , Orosomucoide/química , Orosomucoide/farmacología , Polilisina/análogos & derivados , Polilisina/química , Polilisina/farmacología , ADN , Portadores de Fármacos , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos , Humanos , Plásmidos , Conformación Proteica , Células Tumorales CultivadasRESUMEN
The biosynthesis of HNK-1 carbohydrate is mainly regulated by two glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase (HNK-1 ST). To determine how the two glucuronyltransferases are involved in the biosynthesis of the HNK-1 carbohydrate, we prepared soluble forms of GlcAT-P and GlcAT-S fused with the IgG-binding domain of protein A and then compared the enzymatic properties of the two enzymes. Both GlcAT-P and GlcAT-S transferred glucuronic acid (GlcA) not only to a glycoprotein acceptor, asialoorosomucoid (ASOR), but also to a glycolipid acceptor, paragloboside. The activity of GlcAT-P toward ASOR was enhanced fivefold in the presence of sphingomyelin, but there were no effects on that of GlcAT-S. The activities of the two enzymes toward paragloboside were only detected in the presence of phospholipids such as phosphatidylinositol. Kinetic analysis revealed that the K(m) value of GlcAT-P for ASOR was 10 times lower than that for paragloboside. Furthermore, acceptor specificity analysis involving various oligosaccarides revealed that GlcAT-P specifically recognized N-acetyllactosamine (Galbeta1-4GlcNAc) at the nonreducing terminals of acceptor substrates. In contrast, GlcAT-S recognized not only the terminal Galbeta1-4GlcNAc structure but also the Galbeta1-3GlcNAc structure and showed the highest activity toward triantennary N-linked oligosaccharides. GlcAT-P transferred GlcA to NCAM about twice as much as to ASOR, whereas GlcAT-S did not show any activity toward NCAM. These lines of evidence indicate that these two enzymes have significantly different acceptor specificities, suggesting that they may synthesize functionally and structurally different HNK-1 carbohydrates in the nervous system.