Biomedical subjects

A Hille

Publications and source records attributed to A Hille.

At least 19 recordsLinked to original sources

PubMed1995

Comparative localization of mannose-6-phosphate receptor with 2,6sialyltransferase in HepG2 cells: an analysis by confocal double immunofluorescence microscopy.

Recent advances in confocal immunofluorescent microscopy have led to significant improvements in delineating membrane-bounded organelles. In this study using HepG2 cells we focused on two functionally distinct but closely apposed organelles that have been difficult to distinguish by conventional immunofluorescent microscopy, namely the Golgi apparatus, the trans Golgi network (TGN) and late endosomes. The following markers were used: for the Golgi apparatus beta 1,4galactosyltransferase (gal-T), for the TGN, 2, 6(N)sialytransferase (sia-T) and for late endosomes/TGN, the mannose-6-phosphate/insulin growth factor II receptor (CIMPR). In addition, that part of the TGN previously shown to contain CIMPR was also identified using antibodies to the gamma-chain of the HA-1 adaptor (Klumperman et al. J. Cell Biol. 121, 997-1010 (1993)). True colocalization of intracellular antigens was ascertained by double staining of gal-T using both monoclonal and polyclonal antibodies. As previously reported, our results revealed essentially complete colocalization of gal-T and sia-T in this cell line. While the compartments containing CIMPR appeared to overlap with those containing sia-T by conventional immunofluorescence, both compartments were clearly distinct by double-label confocal microscopy. Differences between these organelles became more evident following treatment with brefeldin A. Finally, HA-1 gamma-chain was also localized to structures that were close to but clearly different from the sia-T-containing compartment. Absence of colocalization of CIMPR or HA-1 gamma-chain with sia-T indicates that these markers are enriched in distinct domains of the trans Golgi network.

Adaptor Protein Complex gamma Subunits↗

PubMed1993

Expression of Epstein-Barr virus nuclear antigen 1,2A and 2B in the baculovirus expression system: serological evaluation of human antibodies to these proteins.

The Epstein-Barr virus encoded nuclear antigens 1,2A, and 2B (EBNA 1, EBNA 2A, and EBNA 2B) were expressed in a baculovirus system. The full length recombinant proteins were recognized by polyclonal rabbit sera and by human sera. An immunofluorescence (IF) test for the differentiation between EBNA 1 and EBNA 2 antibodies in human sera was established with the expressed proteins. None of 55 sera of patients with infectious mononucleosis (IM) had anti-EBNA 1 antibodies while 36 of these sera had anti-EBNA 2A antibodies. Conversely, 47 of 51 sera from EBV-positive healthy carriers had anti-EBNA 1 antibodies and 18 of these sera had anti-EBNA 2A antibodies. The sensitivity and specificity of the EBNA 1 IF for the diagnosis of IM were higher as compared to conventional anti-complement immunofluorescence (ACIF). In the IF test differentiation between type A and type B EBV infection was only possible in sera from the IM patients. An immunoblot (IB) with low amounts of baculovirus expressed EBNA 2A and EBNA 2B antigen was carried out. Twenty-nine of 31 sera from IM patients or from healthy carriers with EBNA 2 antibodies reacted predominantly with EBNA 2A, whereas a known type B serum reacted strongly with EBNA 2B than with EBNA 2A.

Adolescent↗

PubMed1993

Immunological detection of proteins associated with the Epstein-Barr virus nuclear antigen 2A.

The Epstein-Barr virus nuclear antigen 2A (EBNA-2A) has been strongly implicated in the EBV-mediated B-cell transformation process. Since EBNA-2A might exert this function through interaction with proteins of the infected cell, we studied the association of EBNA-2A with cellular proteins. Immunoprecipitation of EBNA-2A from 32P-labeled cell extracts separated by sucrose gradient centrifugation revealed the presence of phosphoproteins complexed with the two forms of the EBNA-2A sedimenting at 13 S and 34 S. Prominent bands were observed at 250, 170, 120, 110, 105, and 95 kDa with minor species at 78, 52, 45, 31, 26, 22 and 18 kDa. By "West-Western" or "Far-Western" blotting using EBNA-2A protein from insect cells as a probe we detected binding to proteins migrating with apparent molecular masses of about 200, 130, 110, 105, 95, and 31 kDa with minor species detectable at 90, 68, 50-55, 40, and 17 kDa. The protein with an apparent molecular mass of 31 kDa was identified by competition experiments as histone H1. Some of the EBNA-2A-complexed phosphoproteins, notably the proteins of 110 and 95 kDa, comigrated with the proteins detectable by "West-Western" analysis. The binding of EBNA-2A to the 130-kDa protein was stable against up to 1.5 M NaCl and could not be competed with histone H1. In a similar experiment, the less transforming EBNA-2B which is encoded by the subtype 2 virus bound to most of the proteins detected with EBNA-2A but with strongly reduced efficiency to the protein of 130 kDa indicating that this protein might be a target for EBNA-2 during EBV-mediated transformation.

Animals↗

PubMed1993

Differences in the endosomal distributions of the two mannose 6-phosphate receptors.

Multiple immunolabeling of cryosections was performed to compare the subcellular distributions of the two mannose 6-phosphate receptors (MPRs) involved in the intracellular targeting of lysosomal enzymes: the cation-dependent (CD) and cation-independent (CI) MPR. In two cell types, the human hepatoma cell line HepG2 and BHK cells double transfected with cDNA's encoding for the human CD-MPR and CI-MPR, we found the two receptors at the same sites: the trans-Golgi reticulum (TGR), endosomes, electron-dense cytoplasmic vesicles, and the plasma membrane. In the TGR the two receptors colocalized and were concentrated to the same extent in the same HA I-adaptor positive coated buds and vesicles. Endosomes were identified by the presence of exogenous tracers. The two MPR codistributed to the same endosomes, but semiquantitative analysis showed a relative enrichment of the CI-MPR in endosomes containing many internal vesicles. Two endosomal subcompartments were discerned, the central vacuole and the associated tubules and vesicles (ATV). We found an enrichment of CD-MPR over CI-MPR in the ATV. Lateral segregation of the two receptors within the plane of membranes was also detected on isolated organelles. Double immunolabeling for the CD-MPR and the asialoglycoprotein receptor, which mainly recycles between endosomes and the plasma membrane, revealed that these two receptors were concentrated in different subpopulations of endosomal ATV. The small GTP-binding protein rab4, which has been shown to mediate recycling from endosomes to the plasma membrane, was localized at the cytosolic face of many endosomal ATV. Quantitative analysis of double-immunolabeled cells revealed only a limited codistribution of the MPRs and rab4 in ATV. These data suggest that the two MPRs exit the TGR via the same coated vesicles, but that upon arrival in the endosomes CD-MPR is more rapidly than CI-MPR, segregated into ATV which probably are destined to recycle MPRs to TGR.

Animals↗

PubMed1992

Lysosomal acid phosphatase is internalized via clathrin-coated pits.

The presence of lysosomal acid phosphatase (LAP) in coated pits at the plasma membrane was investigated by immunocytochemistry in thymidine kinase negative mouse L-cells (Ltk-) and baby hamster kidney (BHK) cells overexpressing human LAP (Ltk-LAP and BHK-LAP cells). Double immunogold labeling showed that at various stages of invaginating coated pits LAP colocalized with clathrin and plasma membrane adaptors (HA-2 adaptors). Quantitation of the immunogold label showed similar density of wild-type LAP in coated over non-coated areas of the plasma membrane, whereas an internalization-deficient, truncated mutant of LAP which lacks the cytoplasmic tail was less efficiently included into coated pits. Internalization of anti-LAP antibodies into endosomal vesicles was accompanied by rapid dissociation of the coat proteins as shown by an immunofluorescence assay. The role of clathrin-coated vesicles in internalization of LAP was further corroborated by microinjecting monoclonal antibodies against clathrin or HA-2 adaptors into BHK-LAP cells. Internalization of LAP as detected by an immunofluorescence assay was transiently blocked by microinjected antibodies against clathrin or HA-2 adaptors, whereas unrelated antibodies did not affect internalization. These data suggest that LAP is included into clathrin-coated pits of the plasma membrane for rapid internalization.

Acid Phosphatase↗

PubMed1991

Antibodies against the cytoplasmic tail can differentiate between the quaternary forms of the Mr 46,000 mannose 6-phosphate receptor.

An antiserum against a peptide of the cytoplasmic tail of the Mr 46,000 mannose 6-phosphate receptor is described which recognizes preferentially the tetrameric versus the dimeric form of this receptor. This indicates that the conformation of the cytoplasmic tail, which harbours signals necessary for the trafficking of the receptor, depends on the quaternary structure of the receptor.

Antibodies↗

PubMed1991

Glycosylation of the Mr 46,000 mannose 6-phosphate receptor. Effect on ligand binding, stability, and conformation.

Using site-directed mutagenesis the N-glycosylation sites of the Mr 46,000 mannose 6-phosphate receptor (MPR 46) were identified as asparagine residues 57, 83, 107, and 113. The two outer asparagines carry high mannose-type and the two inner asparagines carry complex-type oligosaccharides. The glycosylation mutants were analyzed for stability, binding activity, and subcellular distribution. Replacing asparagine 57, 83, or 107 by threonine decreased only the stability of the receptor. Replacing asparagine 113 by threonine decreased the stability and binding activity. Deletion of three or all four N-glycosylation sites led in addition to an accumulation of the mutant receptors in endoplasmic reticulum-like structures. Nonglycosylated MPR 46 synthesized in the presence of tunicamycin, thus preserving the asparagine residues, had a normal stability and high affinity binding. The decreased stability and binding activity of the receptor mutants is therefore due to the exchange of asparagine residues rather than to the loss of N-linked oligosaccharides. The nonglycosylated receptor, however, displayed a decreased conformational stability after solubilization as a single cycle of freezing and thawing reduced the binding activity to one-third of the control. Simultaneously, the receptor lost its quaternary structure. It is concluded from these results that the N-glycosylation of the receptor is required for the stability of a high affinity conformation, but not for the binding itself or the intracellular stability.

Cell Compartmentation↗

PubMed1991

Quantitation of Mr 46000 and Mr 300000 mannose 6-phosphate receptors in human cells and tissues.

A highly specific enzyme-linked immunosorbent assay for quantitation of the Mr 46000 Da and Mr 300000 Da mannose 6-phosphate receptors was developed. The assay allows to detect ng amounts of human mannose 6-phosphate receptors. Analysis of human cells and tissues revealed significant differences in their contents of the two mannose 6-phosphate receptors, normalized for total cell protein. The ratio of the two mannose 6-phosphate receptors also differed among cells and tissues, suggesting that their steady state concentrations are regulated independently.

Cell Line↗

PubMed1991

[Polyvalent immunoglobulins in prevention of viral infections in children with neoplastic diseases treated with immunosuppressive therapy].

31 children with leukemias, lymphomas and solid tumors treated by polychemotherapy received prophylactic i.v. polyvalent immunoglobulins during 48 hours after exposion with varicella-zoster-virus, rubella-virus or mumps-virus. None of the 23 children with immunity against the exposed virus developed the infection. 8 children had no antibodies against the exposed virus. The prophylaxis with the polyvalent immunoglobulin protected them against chickenpox (6 children) and rubella (2 children). All children showed an elevation of the IgG level in plasma and the antibody titers against measles-, mumps-, rubella- and varicella-zoster-virus after application of the immunoglobulin. A study with a larger number of patients is necessary to confirm our results, that polyvalent immunoglobulins with high titers against exposed viruses are effective in prevention of this diseases in immunocompromised children.

Adolescent↗

PubMed1991

Endogenous alkaloids in man--synthesis, analytics, in vivo identification, and medicinal importance.

The in vitro and in vivo condensation of endogenous (or administered) reactive amines and carbonyl compounds is reported, leading to alkaloid-type heterocycles. The spectrum of the presented "mammalian alkaloids" ranges from isoquinoline derivatives, via beta-carbolines, through to thiazolidines, arising from vitamin B6, chloral, and glyoxylic acid, respectively. Formation of these compounds may occur accidentally, but might also be induced intentionally, as a therapy for metabolic diseases.

Alkaloids↗

PubMed1990

Occurrence of tyrosine sulfate in proteins--a balance sheet. 1. Secretory and lysosomal proteins.

1. The abundance of tyrosine sulfate in secretory proteins and in various classes of cellular proteins has been quantified and compared to protein-bound carbohydrate sulfate. 2. HepG2 cells and fibroblasts, two cell types showing only the constitutive pathway of secretion, and PC12 cells, which show both the constitutive and the regulated pathway of secretion, were subjected to pulse-chase and/or long-term labelling with [35S]sulfate and [3H]tyrosine, followed by analysis of proteins in the cells and medium. Under both conditions of labelling, 65-92% of the protein-bound tyrosine sulfate and 44-84% of the protein-bound carbohydrate sulfate were found to be secretory. In HepG2 cells, the frequency of sulfation of tyrosine residues, which can be determined independently from protein abundance and the rate of protein synthesis, was 8-22 times higher in proteins secreted into the medium than in cellular proteins. 3. All cell lines studied contained significant amounts, not only of carbohydrate sulfate, but also of tyrosine sulfate in specific cellular proteins. As shown for fibroblasts, these tyrosine-sulfated proteins were retained within the cells for at least 100 min of chase following a pulse with [35S]sulfate and were almost completely recovered in a light membrane fraction after subcellular fractionation. 4. Lysosomes were found to contain small, but significant, amounts of protein-bound tyrosine sulfate in addition to protein-bound carbohydrate sulfate. Protein-bound tyrosine sulfate in lysosomes reached a peak at 20 min of chase and rapidly disappeared thereafter, whereas protein-bound carbohydrate sulfate accumulated after 20 min of chase. Examination of the known sequences of eleven lysosomal enzymes revealed the presence of potential tyrosine sulfation sites in five of them. 5. Our results show that secretory proteins are the most abundant, but not exclusive, in vivo substrates for tyrosine sulfation and suggest the presence of soluble tyrosine-sulfated proteins in lysosomes and other, as yet unidentified, organelles of the secretory pathway. In the following paper in this journal we describe the abundance of tyrosine sulfate in integral membrane proteins.

Amino Acid Sequence↗

PubMed1990

Occurrence of tyrosine sulfate in proteins--a balance sheet. 2. Membrane proteins.

1. The abundance of tyrosine sulfate in membrane proteins was quantified in four different cell lines and compared to that in soluble cellular and secreted proteins. 2. Upon metabolic labelling of HepG2, Ltk-, AtT20 and PC12 cells with [35S]sulfate or [3H]tyrosine, a fraction enriched in integral membrane proteins was found to contain small, but significant, amounts of protein-bound tyrosine sulfate (up to 2.5% of the total cellular plus secreted protein-bound tyrosine sulfate). On the other hand, the frequency of sulfation of tyrosine residues of membrane proteins was within the same order of magnitude as that of secreted proteins, indicating that the low abundance of tyrosine sulfate in membrane proteins was largely a reflection of the low abundance of these proteins themselves. Consistent with this conclusion were the results of an analysis showing that 14 out of 32 selected membrane-spanning proteins contain potential tyrosine sulfation sites. 3. In HepG2 cells, three tyrosine-sulfated integral membrane glycoproteins of molecular mass 100, 125 and 150 kDa were identified. Characterization of the 150-kDa tyrosine-sulfated membrane protein revealed that it was protected from proteolysis in intact cells, suggesting a localization in an intracellular organelle. 4. Together with the results reported in the preceding paper in this journal, our data suggest that tyrosine sulfation occurs in various classes of trans-Golgi-derived proteins, soluble as well as membrane, and extracellularly exposed as well as intracellularly retained, proteins. This suggests that tyrosine sulfation may have a variety of physiological functions, depending on the individual tyrosine-sulfated protein or protein class.

Amino Acid Sequence↗

PubMed1990

Quaternary structure of the Mr 46,000 mannose 6-phosphate specific receptor: effect of ligand, pH, and receptor concentration on the equilibrium between dimeric and tetrameric receptor forms.

The Mr 46,000 mannose 6-phosphate specific receptor exists in solution as a mixture of noncovalently associated dimeric and tetrameric forms. The two quaternary forms were separated by sucrose density centrifugation, and their composition was assessed by cross-linking with bifunctional reagents followed by SDS-polyacrylamide gel electrophoresis. The dependence of equilibrium between the dimeric and tetrameric forms on pH, receptor concentration, and presence of mannose 6-phosphate was studied. The formation of tetrameric forms is favored by pH values around 7, high receptor concentration, and presence of mannose 6-phosphate ligand. Tetrameric forms bind stronger at pH 7 to phosphomannan-Sepharose 4B than dimeric forms. Both quaternary forms dissociate at the same pH from a mannose 6-phosphate affinity matrix. When starting with dimeric or tetrameric forms, the equilibrium between dimeric and tetrameric forms is reached at pH 7.5 and 4 degrees C after 6-8 days. The presence of 5 mM mannose 6-phosphate shifts the equilibrium toward tetrameric forms. At pH 4.5 and 4 degrees C, the association of dimeric to tetrameric forms is negligible, while tetrameric forms dissociate to dimeric forms within 12 h. The results demonstrate that oligomerization is an intrinsic property of MPR-46 that is affected by ligand binding, pH, and receptor concentration.

Carrier Proteins↗

PubMed1990

Endogenous alkaloids in man, VII: 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline--a potential chloral-derived indol alkaloid in man.

The trichloromethyl tetrahydro-beta-carboline 5, an imaginable, chloral-derived mammalian indol "alkaloid", was prepared in high yields and was shown to be formed even under mild, physiological conditions, in aqueous medium. For its detection in low concentrations, a chromatographic procedure was elaborated. Furthermore, its potential metabolite 8 was synthesized for the first time.

Alkaloids↗

PubMed1990

Mannose 6-phosphate receptor dependent secretion of lysosomal enzymes.

BHK and mouse L cells transfected with the cDNA for the human 46 kd mannose 6-phosphate receptor (MPR 46) secrete excessive amounts of newly synthesized mannose 6-phosphate containing polypeptides. The secretion is dependent on the amount, the recycling and the affinity for ligands of MPR 46. Incubation of transfected cells with antibodies blocking the binding site of MPR 46 reduces the secretion, and cotransfection with the cDNA for the human 300 kd mannose 6-phosphate (MPR 300) restores it to normal values. These results indicate that the two mannose 6-phosphate receptors compete for binding of newly synthesized ligands. In contrast to ligands bound to MPR 300, those bound to the MPR 46 are transported to and released at a site, e.g. early endosomes or plasma membrane, from where they can exit into the medium. Since antibodies blocking the binding site of MPR 46 reduce secretion also in non-transfected BHK and mouse L cells, at least part of the basal secretion of M6P-containing polypeptides is mediated by the endogenous MPR 46.

Animals↗

PubMed1990

Assembly of the ligand-binding conformation of Mr 46,000 mannose 6-phosphate-specific receptor takes place before reaching the Golgi complex.

The early steps in the biosynthesis of Mr 46,000 mannose 6-phosphate-specific receptor (MPR 46) have been studied by in vivo labeling of transfected BHK cells. The acquisition of phosphomannan-binding activity was compared with changes in protein structure and posttranslational modifications of MPR 46. Intramolecular disulfide bonds were formed before MPR 46 acquired a ligand-binding conformation. A conformational change that resulted in increased trypsin resistance, formation of highly immunogenic epitopes and assembly to noncovalently linked homodimers was observed almost simultaneously with the acquisition of ligand-binding activity. MPR 46 was shown to acquire ligand-binding activity before N-linked oligosaccharides were processed to complex-type forms. Maturation of the ligand-binding conformation was observed under conditions where transport to the Golgi was blocked by lowering the temperature to 16 degrees C, or by addition of brefeldin A or dinitrophenol to the medium at 37 degrees C. This suggests that receptor maturation and assembly take place before reaching the Golgi complex. The affinity towards phosphomannan-containing ligands was shown to be similar for the high-mannose and complex-glycosylated forms of MPR 46.

Animals↗

PubMed1990

Regulation of chromogranin B/secretogranin I and secretogranin II storage in GH4C1 cells.

GH4C1 cells are a rat pituitary tumor cell strain in which the level of cellular prolactin (PRL) and PRL-containing secretory granules can be regulated by hormone treatment. The chromogranins/secretogranins (Sg) are a family of secretory proteins which are widely distributed in the secretory granules of endocrine and neuronal cells. In the present study, we investigated in GH4C1 cell cultures the regulation of the cell content of the Sg by immunoblotting and the relationship between the storage of Sg I and Sg II and PRL by double immunocytochemistry. GH4C1 cells grown in the presence of gelded horse serum, a condition in which these cells contain a low level of secretory granules, contained low levels of PRL, Sg I, and Sg II. Treatment of GH4C1 cells with a combination of 17 beta-estradiol, insulin, and epidermal growth factor for 3 days, known to induce a marked increase in the number of secretory granules, increased the cell contents of PRL, Sg I, and Sg II. To determine whether the induction of PRL was morphologically associated with that of the Sg, the distribution of PRL and the Sg was determined by double immunofluorescence microscopy. After hormone treatment, 54% of cells showed positive PRL immunoreactivity, fluorescence being extranuclear and consistent with staining of the Golgi zone and secretory granules. Forty-six percent of PRL-positive cells stained coincidently for Sg I, while 72% of the PRL cells were also reactive with anti-Sg II. To determine whether PRL storage was associated with storage of at least one of the Sg, cells were stained with anti-PRL and anti-Sg I and anti-Sg II together. Eighty-six percent of PRL cells stained for one or the other of the Sg. Therefore, PRL storage in GH4C1 cell cultures is closely but not completely associated with the storage of Sg I and/or II.

Animals↗

PubMed1989

Sulfation of two tyrosine-residues in human complement S-protein (vitronectin).

Human S-protein (vitronectin) and hemopexin, two structurally related plasma proteins of similar molecular mass and abundance, were analyzed for tyrosine sulfation. Both proteins were synthesized and secreted by the human hepatoma-derived cell line Hep G2, as shown by immunoprecipitation from the culture medium of [35S]methionine-labelled cells. When Hep G2 cells were labelled with [35S]sulfate, S-protein, but not hemopexin, was found to be sulfated. Half of the [35S]sulfate incorporated into S-protein was recovered as tyrosine sulfate. The stoichiometry of tyrosine sulfation was approximately two mol tyrosine sulfate/mol S-protein. Examination of the S-protein sequence for the presence of the known consensus features for tyrosine sulfation revealed three potential sulfation sites at positions 56, 59 and 401. Tyrosine 56 is the most probable site for stoichiometric sulfation, followed by tyrosine 59 which appears more likely to become sulfated than tyrosine 401. Tyrosines 56 and 59 are located in the anionic region of S-protein which has no homologous counterpart in hemopexin. We discuss the possibility that tyrosine sulfation of the anionic region of S-protein may stabilize the conformation of S-protein in the absence of thrombin-antithrombin III complexes and may play a role in its binding to thrombin-antithrombin III complexes during coagulation.

Amino Acid Sequence↗