RESUMO
Based on sequence homologies with leguminous lectins, the intermediate compartment marker ERGIC-53 was proposed to be a member of a putative new class of animal lectins associated with the secretory pathway. Independent, a promyelocytic protein, MR60, was purified by mannose-column chromatography, and a cDNA was isolated that matched MR60 peptide sequences. This cDNA was identical to that of ERGIC-53 and homologies with the animal lectin family of the galectins were noticed. Not all peptide sequences of MR60, however, were found in ERGIC-53, raising the possibility that another protein associated with ERGIC-53 may possess the lectin activity. Here, we provide the first direct evidence for a lectin function of ERGIC-53. Overexpressed ERGIC-53 binds to a mannose column in a calcium-dependent manner and also co-stains with mannosylated neoglycoprotein in a morphological binding assay. By using a sequential elution protocol we show that ERGIC-53 has selectivity for mannose and low affinity for glucose and GlcNAc, but no affinity for galactose. To experimentally address the putative homology of ERGIC-53 to leguminous lectins, a highly conserved protein family with an invariant asparagine essential for carbohydrate binding, we substituted the corresponding asparagine in ERGIC-53. This mutation, as well as a mutation affecting a second site in the putative carbohydrate recognition domain, abolished mannose-column binding and co-staining with mannosylated neoglycoprotein. These findings establish ERGIC-53 as a lectin and provide functional evidence for its relationship to leguminous lectins. Based on its monosaccharide specificity, domain organization, and recycling properties, we propose ERGIC-53 to function as a sorting receptor for glyco-proteins in the early secretory pathway.
Assuntos
Cálcio/metabolismo , Lectinas de Ligação a Manose , Manose/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Transformada , Chlorocebus aethiops , Epitopos , Expressão Gênica , Humanos , Lectinas/química , Manose/farmacologia , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , Relação Estrutura-AtividadeRESUMO
We have designed histidylated oligolysines which increase the uptake, the cytosolic delivery and the nuclear accumulation of antisense oligonucleotides (ODN). Flow cytometry analysis showed a 10-fold enhancement of the ODN uptake in the presence of histidylated oligolysines. The intracellular localizations of fluorescein-labeled ODN and of rhodamine-labeled histidylated oligolysines were investigated by confocal microscopy. Histidylated oligolysines favor the cyto-solic delivery of ODN from endosomes and increase their nuclear accumulation. In contrast, in their absence fluorescent ODN were not observed inside the nucleus but were distributed overwhelmingly within the vesicles in the cytosol. In addition, histidylated oligolysines yielded a more than 20-fold enhancement of the biological activity of antisense ODN towards the inhibition of transient as well as constitutive gene expression. Prevention of endosome lumen acidification using bafilomycin A(1)abolished the effect of histidylated oligolysines, suggesting that protonation of the histidyl residues was involved in the transmembrane passage of ODN.
Assuntos
Histidina/química , Lisina/metabolismo , Oligodesoxirribonucleotídeos Antissenso/metabolismo , Tionucleotídeos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Membrana Celular/metabolismo , Citometria de Fluxo , Humanos , Concentração de Íons de Hidrogênio , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Lisina/química , Microscopia Confocal , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tionucleotídeos/farmacologia , Células Tumorais CultivadasRESUMO
The low and unpredictable uptake and cytosolic transfer of oligonucleotides (ODN) is a major reason for their limited benefit. Improving the ODN potential for therapy and research requires a better understanding of their receptor-mediated endocytosis. We have undertaken to identify a membrane ODN receptor on HepG2 cells by ligand blotting of cell extracts with [(125)I]ODN and by photolabelling of living cells with a [(125)I]ODN-benzophenone conjugate. A major band at 66 kDa was identified by the two methods. Its labelling was saturable and competed for by unlabelled ODN of various sequences and irrespective of the presence of a phosphodiester or phosphoro-thioate backbone. This protein remained sedimentable after carbonate extraction, indicating strong membrane association. About half of the total cell amount resisted extensive surface proteolysis, suggesting a dual localisation at the plasma membrane and cytoplasmic vesicles. The protein was purified using a biotinylated ODN-benzophenone conjugate by photocrosslinking followed by streptavidin affinity purification. A sequence obtained by Edman degradation showed no homology with known proteins. Using anti-peptide antisera, labelling by western blotting revealed at 66 kDa a band with comparable properties as found by ligand blotting. Thus, a new membrane protein acting as an ODN receptor has been demonstrated.
Assuntos
Oligodesoxirribonucleotídeos/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Bases , Western Blotting , Humanos , Marcadores de Fotoafinidade , Ligação Proteica , Receptores de Superfície Celular/isolamento & purificação , Frações Subcelulares , Células Tumorais CultivadasRESUMO
A new deprotection procedure enables a medium scale preparation of phosphodiester and phosphor-othioate oligonucleotides substituted with a protected thiol function at their 5'-ends and an amino group at their 3'-ends in good yield (up to 72 OD units/micromol for a 19mer phosphorothioate). Syntheses of 3'-amino-substituted oligonucleotides were carried out on a modified support. A linker containing the thioacetyl moiety was manually coupled in two steps by first adding its phosphor-amidite derivative in the presence of tetrazole followed by either oxidation or sulfurization to afford the bis-derivatized oligonucleotide bound to the support. Deprotection was achieved by treating the fully protected oligonucleotide with a mixture of 2,2'-dithiodipyridine and concentrated aqueous ammonia in the presence of phenol and methanol. This proced-ure enables (i) cleavage of the oligonucleotide from the support, releasing the oligonucleotide with a free amino group at its 3'-end, (ii) deprotection of the phosphate groups and the amino functions of the nucleic bases, as well as (iii) transformation of the 5'-terminal S -acetyl function into a dithiopyridyl group. The bis-derivatized phosphorothioate oligomer was further substituted through a two-step procedure: first, the 3'-amino group was reacted with fluorescein isothiocyanate to yield a fluoresceinylated oligo-nucleotide; the 5'-dithio-pyridyl group was then -quantitatively reduced to give a free thiol group which was then substituted by reaction with an N alpha-bromoacetyl derivative of a signal peptide containing a KDEL sequence to afford a fluoresceinylated peptide-oligonucleotide conjugate.
Assuntos
Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/síntese química , Sinais Direcionadores de Proteínas , Tionucleotídeos/química , Tionucleotídeos/síntese química , Amidas/química , Aminas/química , Sequência de Aminoácidos , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Fluoresceína-5-Isotiocianato/química , Espectrometria de Massas , Metanol/química , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/isolamento & purificação , Oligopeptídeos/química , Oligopeptídeos/genética , Oxirredução , Fenol/química , Fosfatos/química , Fosforamidas , Ácidos Fosfóricos/química , Piridinas/química , Compostos de Sulfidrila/química , Tetrazóis/química , Tionucleotídeos/genéticaRESUMO
Liver cancer is one of the most frequent and lethal malignancies worldwide. Early detection is hampered by the absence of reliable markers. Mice transgenic for the SV40 large T antigen under the control of a liver-specific promoter spontaneously develop well-differentiated hepatocellular carcinomas between 8 to 10 weeks of age. They are excellent models to investigate the alterations of protein expression in the early stages of tumor development and to follow these changes during tumor progression. In the present study, we analyzed the glycosylation changes occurring during tumor development in transgenic mice expressing the SV40 T antigen under the control of the antithrombin III promoter. The analysis of serum and liver glycoproteins by an ELISA type assay, using the lectin from Sambucus nigra (SNA) as a probe, revealed the presence of increased levels of Neu5Ac alpha2,6Gal beta1,4GlcNAc on N-glycans in the tumor-bearing transgenic mice as compared to controls. On serum glycoproteins the increase in alpha2,6 sialylation followed tumor progression, reaching up to 10 times control levels. However, significantly higher SNA binding (2-fold) could already be observed on serum glycoproteins from mice exhibiting only microscopically small neoplastic foci. On liver membrane glycoproteins, the increase in alpha2,6 sialylation was less pronounced, reaching two to three times control values in 6-month-old mice. Western blotting of serum and liver proteins with radiolabeled SNA showed that all glycoproteins that bind the lectin in controls exhibit larger amounts of Neu5Ac alpha2,6Gal beta1,4GlcNAc on N-glycans in the tumor-bearing mice. This general increase in alpha2,6 sialylation on all glycoproteins is due to the increased activity of the galactoside:alpha2,6 sialyltransferase (ST6Gal I), which specifically transfers Neu5Ac residues in alpha2,6 linkage to Gal beta1,4GlcNAc units on N-glycans. As for the structures synthesized by the enzyme, the increase of ST6Gal I activity in the serum as well as in liver microsomes of the transgenic mice followed tumor progression. Interestingly, the activity of the galactoside:alpha2,3 sialyltransferase (ST3Gal III), which uses the same acceptor substrate (Gal beta1,4GlcNAc), was unchanged in the earlier stages of tumor development but decreased in the serum and in liver microsomes from later stages. Using a rat ST6Gal I cDNA as a probe, Northern blots of total RNA extracted from the livers of control and transgenic mice revealed an increased (4-fold) expression of the ST6Gal I gene. The single transcripts detected in both normal and cancerous liver showed identical size.
Assuntos
Neoplasias Hepáticas Experimentais/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Lectinas de Plantas , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Sialiltransferases/metabolismo , Animais , Antígenos Virais de Tumores/fisiologia , Proteínas Sanguíneas/metabolismo , Sequência de Carboidratos , Progressão da Doença , Indução Enzimática , Ensaio de Imunoadsorção Enzimática , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/metabolismo , Glicosilação , Lectinas/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Transgênicos , Microssomos Hepáticos/metabolismo , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Neoplásico/análise , RNA Neoplásico/genética , Ratos , Proteínas Inativadoras de Ribossomos , Sialiltransferases/genética , Vírus 40 dos Símios/genéticaRESUMO
The permeabilization by alpha-helical peptides of nucleated mammalian cells can be monitored by flow cytometry. Ethidium bromide, a non fluorescent and poorly membrane permeant molecule, becomes strongly fluorescent only upon binding to DNA. On this basis, the permeabilization of the plasma membrane of HL60 promyelocytic cells induced by alpha-helical peptides such as melittin, succinylated melittin and anionic peptides derived from the N-terminus of HA2 subunit of the influenza virus hemagglutinin, was measured. Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH2) caused a rapid (< 5 min) and dose-dependent (ED50 = 0.5 microM) permeabilization of HL60 cells at neutral pH, whereas the succinylated derivative induced cell permeabilization only at pH below 4.5 with an ED50 = 18 microM. The permeabilization by the anionic E5CA peptide (GLFEAIAEFIEGGWEGLIEGCA) containing 5 glutamic residues occurred (ED50 = 11 microM) at pH ranging from 6.5 to 6.0; replacing the tryptophan residue in position 14 by a phenylalanine residue decreased by about 1 unit the pH at which membrane permeabilization was effective. The membrane permeabilization activity of the E5CA peptide was reversibly abolished when the peptide was linked to a protein carrier. These results show that alpha-helical peptide-induced membrane permeabilization can be easily monitored by using flow cytometry in the presence of a non permeant dye. This method allows a rapid screening and an efficient mean of selection of peptides suitable to induce membrane permeabilization.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Peptídeos/farmacologia , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Células HL-60 , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas Virais/química , Hemaglutininas Virais/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Meliteno/análogos & derivados , Meliteno/farmacologia , Dados de Sequência Molecular , Neuropeptídeos/química , Neuropeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologiaRESUMO
A polylysine partially substituted with polyhydroxyalkanoyl residues and specially with gluconoyl residues was developed in order to increase the transfection efficiency by decreasing the strength of the electrostatic interactions between the DNA and the cationic polymer. Partially gluconoylated polylysine/DNA complexes were more easily dissociated in solution and their transfection efficiency in the presence of chloroquine, evaluated with HepG2 cells, a human hepatocarcinoma line, was higher when 43 +/- 4% of the epsilon-amino groups of polylysine were blocked with gluconoyl residues. Partially gluconoylated polylysine/plasmid complexes were efficient in transfecting different adherent as well as non-adherent cell lines. Partially gluconoylated polylysine formed highly soluble (above 100 micrograms/ml in DNA) complexes with DNA plasmids. In addition, partially gluconoylated polylysine bearing few lactosyl residues increased the transfection efficiency of HepG2 cells which express a galactose-specific membrane lectin.
Assuntos
Plasmídeos , Polilisina , Transfecção/métodos , Acilação , Sequência de Aminoácidos , Carcinoma Hepatocelular/genética , Cloroquina , DNA Recombinante , Gluconatos , Humanos , Lactonas , Neoplasias Hepáticas/genética , Dados de Sequência Molecular , Fosfatidiletanolaminas , Polilisina/química , Solubilidade , Células Tumorais CultivadasRESUMO
Trifluoroacetamide was found to be a good quencher of tryptophan fluorescence, and the quenching was shown to proceed via both a dynamic and a static process. The respective quenching constants were determined by the measurement of the decrease of the fluorescence lifetime in the presence of the quencher. The static and the bimolecular rate quenching constants of N-acetyltryptophanamide are equal to 0.34 1 X mol-1 and 1.9 X 10(9) 1 X mol-1 X s-1, respectively. These values indicate that trifluoroacetamide is an efficient quencher of tryptophan fluorescence. This conclusion is also supported by a complete quenching of bovine serum albumin and wheat germ agglutinin fluorescence. In the case of lysozyme, trifluoroacetamide quenches the fluorescence of tryptophan residues which fluoresce with a maximum at 348 nm but not the buried tryptophan residues which fluoresce with a maximum at 333 nm. Trifluoroacetamide quenching of wheat germ agglutinin emission confirms the homogeneity and the high accessibility of emitting tryptophan residues, in agreement with a previous report (Privat, J.P. and Monsigny, M. (1975) Eur. J. Biochem. 60, 555-567). The tryptophan fluorescence decay of wheat germ agglutinin is biexponential even in the presence of the quencher; the static and bimolecular rate quenching constants are equal to 0.22 1 X mol-1 and 0.92 X 10(9) 1 X mol-1 X s-1, respectively. In the presence of a specific lectin ligand, the methyldi-N,N'-trifluoroacetyl-beta-chitobioside, the quenching of wheat germ agglutinin fluorescence involves a direct contact between tryptophan residues and trifluoroacetamido groups of the ligand and in contrast with the quenching induced by free trifluoroacetamide shows that the tryptophan fluorescence is not fully quenched.
Assuntos
Fluoracetatos , Proteínas/análise , Ácido Trifluoracético , Triptofano , Acetamidas , Animais , Bovinos , Galinhas , Clara de Ovo , Cinética , Lectinas , Muramidase , Soroalbumina Bovina , Espectrometria de Fluorescência/métodos , Aglutininas do Germe de TrigoRESUMO
Nucleocytoplasmic transport of rat liver mRNA is thought to be regulated by a nucleoside triphosphatase whose activity in the intact nuclear envelope is stimulated by the 3'poly(A) tail of poly(A)+ mRNA. In contrast to the liver mRNA, the mRNA from rat brain contains a great population of poly(A)- mRNA's that does not appear until after birth. Measurements of the nuclear-envelope-associated enzyme activities involved in mRNA transport, and their dependence on endogenous (isolated cytoplasmic mRNA-transport-stimulating proteins) and exogenous (poly(A), lectins, and neoglycoproteins) factors during prenatal and postnatal rat brain and liver development, revealed marked organ-dependent differences paralleling the appearance of the poly(A)- mRNA unique in the brain.
Assuntos
Encéfalo/metabolismo , Fígado/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Poli A/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Animais , Transporte Biológico , Encéfalo/crescimento & desenvolvimento , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Ativação Enzimática/efeitos dos fármacos , Glicoproteínas/farmacologia , Lectinas/farmacologia , Fígado/crescimento & desenvolvimento , Membrana Nuclear/enzimologia , Nucleosídeo-Trifosfatase , Monoéster Fosfórico Hidrolases/metabolismo , Poli A/farmacologia , Ratos , Ratos EndogâmicosRESUMO
Vascular endothelial cell addressins play an important role in lymphocyte homing in secondary lymphoid organs and in chronic inflammatory areas. A SV40 large T antigen-immortalized cell line from peripheral lymph nodes, HECa1O [Bizouarne et al., 1993a], was used to characterize the location of addressins with regard to environmental factors and cytokines. For this purpose, two monoclonal antibodies, MECA 79 and MECA 367, specific for peripheral lymph node vascular addressin and for mucosal addressin (Peyer's patches), respectively, were bound to unstimulated HECa1O cells. Both mucosal and peripheral addressins were detected inside the cells and in cellular extracts of the resting cells. On the cell surface, both addressins could be evidenced on the same cells at a moderate level of expression. They partly mediate the EL4/EL4IL2 lymphoma cells' adhesion to HECa1O cells. Supernatants of cultured peripheral lymph node or Peyers' patch cells induced expression of MECA 79 or MECA 367 antigens, respectively, on the surface of HECa1O cells. Interleukins, IL-7, IL-3, and IL-8, induced the cell-surface appearance of MECA 79 but not of MECA 367 antigen. Therefore, the same cell type synthesizes both antigens, but the expression of these antigens on the cell surface is independently regulated, thus uncovering a characteristic tissue type-specific as well as environment-sensitive properties of microvascular endothelial cells.
Assuntos
Antígenos de Superfície/metabolismo , Endotélio Vascular/imunologia , Imunoglobulinas/metabolismo , Linfonodos/citologia , Mucoproteínas/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Animais , Western Blotting , Adesão Celular , Moléculas de Adesão Celular , Células Cultivadas , Citocinas/farmacologia , Citometria de Fluxo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peso Molecular , Nódulos Linfáticos Agregados/citologiaRESUMO
We have examined the membrane lectin expressed by immortalized normal and cystic fibrosis (CF) airway epithelial cells, using fluorescein-labeled neoglycoproteins; the uptake of plasmid DNA using fluoresceinylated glycoplexes (plasmid/glycosylated polylysine complexes); and the efficiency of gene transfer when glycosylated polylysines and glycosylated, partially gluconoylated polylysines were used as vectors. The most efficient uptake of neoglycoproteins by normal and CF cells was obtained with mannosylated BSA (bovine serum albumin). Similarly, the most efficient uptake of plasmid DNA was obtained with glycoplexes bearing alpha-D-Man residues. Surprisingly, glycoplexes bearing alpha-D-Man residues were poorly efficient for gene transfer into normal and CF cells. The highest luciferase activity was achieved with lactosylated polylysine- and beta-D-GlcNAc-substituted gluconoylated polylysine as vectors. Gene transfer efficiency obtained with gluconoylated polylysine bearing beta-D-GlcNAc residues was similar to that observed with polyethylenimine (PEI; 25 and 800 kDa) and 10-fold higher than that observed with lipofectin and LipofectAMINE. These results suggest that the transfection efficiency with glycoplexes is not determined only by the specificity of the lectin expressed at the cell surface membrane but also by intracellular trafficking of the glycoplexes, which could be mediated by lectins present inside the cells.
Assuntos
Brônquios/metabolismo , Metabolismo dos Carboidratos , Fibrose Cística/terapia , Técnicas de Transferência de Genes , Glicosilação , Traqueia/metabolismo , Linhagem Celular , Sobrevivência Celular , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Citometria de Fluxo , Corantes Fluorescentes , Expressão Gênica , Vetores Genéticos , Glicoproteínas/metabolismo , Humanos , Lipossomos , Microscopia Confocal , Plasmídeos/análise , Polietilenoimina/farmacologia , Polilisina/metabolismo , Fatores de TempoRESUMO
Macrophages are putative target cells for expressing an exogenous gene with therapeutical effects. Knowing that macrophages express membrane lectins mediating endocytosis of their ligands, DNA/glycosylated polylysine complexes were used to transfect human blood monocyte-derived macrophages. Monocytes from human peripheral blood were matured in culture for 7 days to differentiate into macrophage-like cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Adherent cells, which displayed characteristic macrophage markers, CD 14, CD 11b, HLA-DR, and HLA-ABC antigens and mannose receptor, were transfected by DNA/glycosylated polylysine complexes in the presence of chloroquine. The luciferase reporter gene expression was maximal 24 hr after transfection with a DNA/mannosylated polylysine complex and by using plasmids in which the promoters (either the long terminal repeat of the human immunodeficiency virus or the human cytomegalovirus) drove the luciferase gene expression. Luciferase gene expression was lower when the promoter was the early region of the large T antigen of SV40 virus. Transfection mediated by DNA/mannosylated polylysine complexes was much more efficient than with DEAE-dextran or lipofectin. The possibility of transferring and expressing an exogenous gene into macrophage-like cells by using a nonimmunogenic synthetic vector as a DNA carrier opens new ways to develop nonviral gene therapy strategies.
Assuntos
DNA , Técnicas de Transferência de Genes , Macrófagos/fisiologia , Polilisina , Contagem de Células , Cloroquina/farmacologia , Dextranos , Expressão Gênica , Glicosilação , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-3/farmacologia , Luciferases/genética , Monócitos/fisiologia , Plasmídeos , Regiões Promotoras Genéticas , Fatores de Tempo , TransfecçãoRESUMO
To provide an alternative to viral vectors for the transfer of genes into airway epithelial cells in cystic fibrosis (CF), a novel set of substituted polylysines were employed. Polylysine was partially neutralized by blocking a number of positively charged residues with gluconoyl groups. In addition, polylysine was substituted with sugar residues on a specified number of amino groups. Using the gluconoylated polylysine as vector, the pCMVLuc plasmid gave high expression of the reporter gene luciferase in immortalized CF/T43 cells. The luciferase activity was 75-fold greater in the presence of 100 microM chloroquine. Luciferase gene expression persisted at high levels for up to at least 120 hr following transfection. Glycosylated polylysines/pCMVLuc complexes were compared to the gluconoylated polylysine/pCMVLuc complex and beta-Gal-, alpha-Glc-, and Lac-substituted polylysines gave 320%, 300%, and 290%, respectively, higher expression of the reporter gene luciferase. Luciferase expression ranged from 35 to 2 ng of luciferase per milligram of cell protein in the order: beta-Gal = alpha-Glc = Lac > alpha-Gal = Rha = Man > beta-GalNAc > alpha-GalNAc = alpha-Fuc, suggesting that the transfection efficiency is sugar dependent. Most importantly, in primary cultures of both CF and non-CF airway epithelial cells grown from tracheal tissue explants, lactosylated polylysine gave uniformly high expression of luciferase. The glycosylated polylysines provide an attractive nonviral approach for the transfer of genes into airway epithelial cells.
Assuntos
Fibrose Cística/terapia , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Polilisina/análogos & derivados , Contagem de Células , Células Cultivadas , Cloroquina/farmacologia , Epitélio/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Genes Reporter/genética , Terapia Genética/métodos , Gluconatos/metabolismo , Glicosilação , Humanos , Lactonas , Lactose/análogos & derivados , Luciferases/genética , Luciferases/metabolismo , Plasmídeos/genética , Traqueia/metabolismo , Transfecção/genéticaRESUMO
The complete coding sequence of the rabbit galectin-3-encoding cDNA (LGALS3) has been cloned in a single step by using RT-PCR and specific human LGALS3 cDNA primers. The putative protein contains three domains with different degrees of homology to other known LGALS3. The homology is high in the C-terminal moiety corresponding to the carbohydrate-binding domain and is relatively low in the N-terminal moiety.
Assuntos
Antígenos de Diferenciação/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Galectina 3 , Humanos , Dados de Sequência Molecular , Coelhos , Alinhamento de SequênciaRESUMO
Galectin-3 is a galactose-specific lectin which has been shown to be involved in several biological functions such as cell growth regulation, cell aggregation and cell differentiation. The partial cloning of the human genomic sequences reveals the presence of a 651 bp intron, 18 bp downstream of the translation initiation site. This intron contains several regulatory elements found in many eukaryotic genes. This sequence, when inserted upstream of a promoter-free luciferase gene, induces the expression of luciferase, demonstrating the promoter activity of the intron upon transfection in human or murine cells. This promoter activity is down-modulated by wild-type p53 but not by a mutated form of p53.
Assuntos
Antígenos de Diferenciação/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Íntrons , Regiões Promotoras Genéticas , Proteína Supressora de Tumor p53/farmacologia , Sequência de Bases , Clonagem Molecular , DNA/química , DNA/genética , Galectina 3 , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , RNA Mensageiro/análise , RNA Mensageiro/química , Sequências Reguladoras de Ácido NucleicoRESUMO
Freshly isolated human monocytes, which do not contain cell-surface mannose-specific receptors, bind mannose 6-phosphate and actively endocytose mannose 6-phosphate-bearing neoglycoproteins (6-P-Man-F-BSA). Three days after isolation, human monocytes endocytose very actively 6-P-Man-F-BSA as well as Man-F-BSA, and the endocytosed neoglycoproteins are rapidly degraded. These results were obtained in quantitative flow cytofluorometry by using a panel of fluoresceinylated sugar-substituted serum albumins (neoglycoproteins). Thus, in contrast to mannose receptors which appear only after maturation, mannose 6-phosphate receptors are already present on freshly isolated human monocytes.
Assuntos
Lectinas/análise , Monócitos/análise , Proteínas de Transporte/metabolismo , Membrana Celular/análise , Endocitose , Humanos , Manosefosfatos/metabolismo , Receptor IGF Tipo 2RESUMO
A fluorogenic substrate for plasmin, CBZ-Gly-Pro-Arg-AEC, has been synthesized and used to develop a new sensitive photometric and fluorometric assay of plasminogen activator activity. The fluorescence intensity of free AEC at 460 nm is about 3 orders of magnitude higher than that of acyl-AEC. The release of AEC from the peptidyl derivative was monitored fluorometrically after extraction of free AEC in ethylacetate. Under such conditions, the Km was 0.16 mM. This method was used to monitor the activity of plasminogen activator synthetized by fibroblastic cells (BHK 21 C 13) either released in the supernatants or cell-associated.
Assuntos
Rim/análise , Ativadores de Plasminogênio/análise , Animais , Carbazóis , Linhagem Celular , Cricetinae , Fibrinolisina/metabolismo , Fibroblastos/análise , Fluorometria , Cinética , Métodos , OligopeptídeosRESUMO
In the rat liver both hepatocytes and macrophages have been shown to express on the surface lectins with similar binding specificity for galactose residues. Functionally the two lectins differ in the uptake of ligands. Whereas the hepatocytes ingest molecules and small particles (less than 10 nm), the macrophages take up particles only. Antisera raised against hepatic galactose-specific receptor failed to react with the macrophage lectin but blocked ligand binding to the hepatocyte only, indicating either a different antigenic structure or membrane localization of the two lectins.
Assuntos
Lectinas/fisiologia , Fígado/metabolismo , Macrófagos/metabolismo , Animais , Galectinas , Masculino , Neuraminidase/metabolismo , Ratos , Ratos Endogâmicos , Formação de RosetaRESUMO
Lectins associated with the bacterial cell surface of Rhizobium lupini strain LL13 were evidenced by erythrocyte agglutination, by aggregation of neoglycoprotein coated beads and by spectrofluorimetry using fluoresceinylated neoglycoproteins. At pH 5.0, a specific binding of the fluorescein-labelled neoglycoprotein bearing alpha-L-fucose was observed. The binding of this labelled neoglycoprotein is a saturable phenomenon and is inhibited by the same unlabelled neoglycoprotein. Extracts of R lupini obtained by disrupting a bacterial pellet through a French press were stabilized at pH 5.6 by gel filtration and purified to homogeneity by affinity chromatography on Agarose A4 substituted with alpha-L-fucose. A protein with a M(r) approximately 19,000 was specifically eluted from this affinity column with L-fucose. Isoelectric focusing of this sample yielded a single band with pI near 6.7. This protein specifically aggregated L-Fuc-BSA-coated microspheres. The results obtained in the present study indicate that we have purified from Rhizobium lupini strain LL13, a L-fucose binding protein as a lectin.
Assuntos
Fucose/química , Rhizobium/química , Testes de Aglutinação , Proteínas de Transporte/isolamento & purificação , Glicoproteínas/isolamento & purificação , Lectinas/isolamento & purificação , MicroesferasRESUMO
In order to induce the production of antibodies specific for small molecules, it is common to link them to a protein. However, when the small molecule is very hydrophobic it is extremely difficult to prepare such a conjugate. Here, we describe a simple way to obtain an antigenic conjugate under controlled conditions: in a first step a very hydrophobic hapten, cholanic acid, is linked to a dye, basilen blue, in organic solvent; in a second step the cholanic acid-basilen blue conjugate is dissolved in phosphate buffered saline and mixed with rabbit polyclonal anti-basilen blue antibodies previously raised in rabbits against basilen blue-key-hole limpet hemocyanin conjugate. Such a complex, which dissociates very slowly, appears to be a good immunogen in mice. Anti-cholanyl residue monoclonal antibodies were produced and characterized.