Biomedical subjects

V Herzog

Publications and source records attributed to V Herzog.

At least 19 recordsLinked to original sources

PubMed1992

Endocytosis of thyroglobulin is not mediated by mannose-6-phosphate receptors in thyrocytes. Evidence for low-affinity-binding sites operating in the uptake of thyroglobulin.

Thyroglobulin, the major secretory product of thyrocytes, is the macromolecular precursor of thyroid hormones. After its synthesis, thyroglobulin follows a complex secretion, storage and recapture pathway to lysosomes. Porcine thyroglobulin was shown to carry the mannose 6-phosphate-(Man6P)-recognition marker on its N-linked glycans. Since the cation-independent Man6P receptor could also be found on the apical plasma membrane of porcine thyrocytes, we examined the significance of the Man6P signal for the transport of thyroglobulin. Here, we present data implying that Man6P receptors are not relevant for endocytosis of thyroglobulin in thyrocytes. Instead, we provide evidence for the existence of specific, low-affinity-binding sites for thyroglobulin on the apical plasma membrane of thyrocytes responsible for endocytosis of thyroglobulin. Binding studies with intact, polar-organized porcine thyrocytes grown on collagen-coated filters revealed cooperative and saturable binding of thyroglobulin to the apical-plasma-membrane domain at relatively high concentrations of thyroglobulin (20 microM). These observations show that low-affinity interactions between thyroglobulin and the apical plasma membrane play a key role in endocytosis of thyroglobulin and hormone formation in the thyroid. The data in this publication have been published as an abstract [Lemansky, P. and Herzog, V. (1991) J. Cell Biol. 115, 261a].

Absorption↗

PubMed1992

Regulation of the cellular transport and compactation of thyroglobulin.

The cellular transport ot thyroglobulin in thyrocytes is a TSH-regulated process and characterized by a bidirectional export-, storage- and recapture pathway. The regulation of single steps in this pathway is unclear, but a possible basis for this transport appeared to be the presence of the lysosomal targetting, signal mannose-6-phosphate (M6P), on porcine thyroglobulin and the detection of the cation-dependent M6P-receptor on the apical cell surface and the Golgi-complex in thyrocytes. The question arose, why thyroglobulin is not directed on an intracellular pathway to lysosomes thereby following the route characteristic of lysosomal enzymes. Recent observations have shown that the affinity of thyroglobulin to the M6P-receptor is very low and the receptor is presumably not effective in thyrocytes to direct thyroglobulin to lysosomes. Instead, a low affinity binding site for thyroglobulin has been found to operate on the surface of thyrocytes. This receptor is specific for thyroglobulin and is saturable at 8-13 mg thyroglobulin per ml (5-9 microM). The highest affinity of thyroglobulin was that to itself which is the basis for the positive cooperation observed during binding of thyroglobulin to thyrocyte membranes. The receptor has a relative molecular mass of 46 kDa but is not identical with the cation-dependent M6P-receptor. Because of the high-thyroglobulin-concentration in the follicle lumen (100-400 mg/ml) it has always been argued that there is no need for the presence of a thyroglobulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Isolation of insoluble secretory product from bovine thyroid: extracellular storage of thyroglobulin in covalently cross-linked form.

Extracellular storage of thyroglobulin (TG) is an important prerequisite for maintaining constant levels of thyroid hormones in vertebrates. Storage of large amounts is made possible by compactation of TG in the follicle lumen with concentrations of at least 100-400 mg/ml. We recently observed that the luminal content from bovine thyroids can be isolated in an intact state and be separated from soluble TG. For this purpose, bovine thyroid tissue was homogenized and subjected to various steps of purification. This procedure resulted in a pellet of single globules measuring 20-120 microns in diameter. Scanning electron microscopy revealed a unique cobblestone-like surface pattern of isolated globules, showing in detail the impressions of the apical plasma membranes of thyrocytes which had formerly surrounded the luminal content before tissue homogenization. Isolated thyroid globules were rapidly digested by trypsin but extremely resistant to various protein solubilization procedures. Homogenization of isolated globules resulted in the release of approximately 3% of total protein, showing that only a minor proportion of TG was loosely incorporated in thyroid globules whereas approximately 22% appeared to be interconnected with the globule matrix by disulfide bridges. Analysis by SDS-gel electrophoresis and immunoblotting confirmed that the protein released by this procedure consisted of TG. The vast majority (approximately 75%) of the globule matrix protein was found to be covalently cross-linked by non-disulfide bonds. TG in isolated globules was highly iodinated (approximately 55 iodine atoms per 12-S TG subunit) suggesting that the covalent nondisulfide cross-linking occurs in part during the iodination of TG and that this process involves the formation of intermolecular dityrosine bridges. Mechanisms must exist which solubilize or disperse the insoluble luminal content prior to endocytosis of TG.

Animals↗

PubMed1991

Transcytosis in thyroid follicle cells: regulation and implications for thyroglobulin transport.

In order to analyze quantitatively the translocation of plasma membrane during endocytosis and transcytosis and the regulation of these processes in thyroid follicle cells, the apical cell surfaces of resting and TSH-stimulated inside-out follicles were labeled with cationized ferritin. Morphometric analyses showed that the rates of endocytosis and transcytosis are TSH-dependent. More interestingly, whereas the effect of TSH on endocytosis was transient (with a maximum at 16 min), the effect on transcytosis continued to increase until the end of the experiment (i.e, 70 min). During 1 h of endocytosis, the fraction of membrane involved in transcytosis increased by a factor 4 upon TSH stimulation, corresponding to about 12% of the internalized apical plasma membrane area. Cooling to 15 degrees C slowed down, but did not block endocytosis entirely, whereas transcytosis and transfer to lysosomes were totally inhibited In order to quantitate transcytosis of thyroglobulin (TG) and to ascertain whether this molecule undergoes cleavage during transcytosis, inside-out follicles were incubated in a medium containing 3H-labeled TG in the presence of TSH; upon washing and reopening of follicles, the luminal fluid containing TG after transcytosis was found to contain about 10% of the total radioactivity taken up by follicle cells. Transcytosed TG proved to be unmodified with respect to its electrophoretic mobility. We conclude that (i) the fraction of transcytosed TG corresponds approximately to the fraction of membrane involved in this process, (ii) TG does not undergo cleavage during transcytosis, (iii) endocytosis and transcytosis are regulated by TSH but differ in their kinetics after stimulation, and (iv) transcytosis is affected by temperature in a similar way as transfer to lysosomes, suggesting the existence of a common gating step for both pathways.

Animals↗

PubMed1991

[Lead concentration in permanent teeth of the population of Erfurt and its surroundings].

In this study 1616 permanent teeth were collected from 1466 stomatological patients, which are living in Erfurt and surroundings. The lead levels were investigated by flame-AAS. Comparisons were need with following parameters: sex, age, living area, vitality of teeth, smoking, caries and filling of teeth. Mean lead level from investigated teeth was 13.2 microgram Pb/g tooth by mean patients-age of 38 years. The results showed that there have not been significant differences between sex and investigated areas of Erfurt and surroundings. Low influence on lead levels in teeth were found in groups when were investigated following parameters: vitality, smoking, caries, filling of teeth. High influence on tooth lead level was connected with the age of patient that is time of exposition. We found as raising factor on 3% tooth lead level per year. It was also found that incisors pick up more lead than molars and premolars, and molars more than premolars. These differences would be depend of food and respiratory air. The general result of this investigations about the consequence of tooth lead levels by the inhabitants of Erfurt point out that there is no danger of chronic lead intoxication by adults.

Adolescent↗

PubMed1989

Classical pathway of complement activation in normal and diseased human glomeruli.

Monoclonal antibodies (mAb) reactive against complement components involved in the classical activation pathway were applied in an indirect immunoperoxidase technique for the histological study of normal and diseased human renal tissues. Prominent staining with antibodies against the C4d fragment was seen in all glomeruli and some renal arteriolar walls. The C4d staining was mesangial with light microscopy, whereas the subendothelial site of the glomerular basement membrane (GBM) also appeared to be positive in immunoelectron microscopy. In similar localization, albeit with distinctly weaker intensity, IgM and C4 binding protein (C4bp) were detected. In kidney biopsies from patients with various types of glomerulonephritis, C4d reactive antibodies stained the glomerular structures in a strong, diffuse or granular pattern in contrast to the more segmental distribution and weaker staining intensity in normal kidney specimens. Increased amounts of C4d, occasionally also of C4b, were paralleled in diseased kidney tissues by distinct deposits of IgM and/or IgG in the presence of C4bp. This study suggests that the C4d fragment in normal human glomeruli is indicative of a continuous, local complement activation via the classical pathway induced by the physiological deposition of IgM-containing immune complexes.

Animals↗

PubMed1989

[Hormone receptors in the inner ear].

Vasopressin binding sites could be clearly demonstrated in the cochlea. Membrane staining was mainly limited to the apical and ciliar membranes of the cochlear and vestibular hair cells, and hence to membranes in which adenylate cyclase activity could not be demonstrated. In addition to V2-vasopressin receptors that mediate hormonal signals by adenylate cyclase activation and cAMP release, in V1-vasopressin-receptors extracellular vasopressin signal is mediated by the breakdown of inositol phosphates and the release of inositol-triphosphates and diacylglycerol. Inositol triphosphates were found to be responsible for the intracellular mobilization of calcium. The localization of vasopressin binding sites at the hair cell membranes, therefore, suggests that vasopressin contributes to the breakdown and release of phospholipid messenger molecules and is thus probably involved in cochlear and vestibular signal transduction.

Animals↗

PubMed1989

Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites.

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.

Amino Acid Sequence↗

PubMed1989

Human placental sterylsulfatase: immunocytochemical and biochemical localization.

Human placental sterylsulfatase was localised in situ by light and electron microscope immunocytochemical techniques as well as in homogenate and tissue extract fractions by enzyme assays. Light microscope observations on frozen sections of term and preterm placenta revealed sterylsulfatase immunoactivity primarily in the syncytiotrophoblast. Electron microscope observations confirmed the light microscope findings; in addition, they showed that the sulfatase is present in the endoplasmic reticulum of endothelial cells, too. In the syncytiotrophoblast, the enzyme was detectable in the cytoplasmic membrane of the nuclear evelope, in the membranes of the rough endoplasmic reticulum, in the plasma membrane with predominant localisation in coated pits, and in the membranes of endosomes and multivesicular bodies; little or no reactivity was detectable over the membranes of the Golgi complex and of lysosomes. Sterylsulfatase immunoactivity was absent in placentas with hereditary sterylsulfatase deficiency. The observations indicate that human placental sterylsulfatase is normally present in the membranes of compartments along the secretory pathway and the endocytic route of cells lining the fetal and maternal blood. Homogenates of normal term placenta as well as membrane vesicle preparations obtained by extraction of trophoblast tissue with isotonic saline were fractionated by differential centrifugation; the fractions were assayed for specific activities of sterylsulfatase and several marker enzymes of cellular topography. In agreement with our immunocytochemical findings, the results of these biochemical localisation experiments indicate the repeatedly described association of the placental sterylsulfatase with microsomal membranes but also point to the presence of the enzyme's activity in the microvillous plasma membrane of the syncytiotrophoblast. This localisation of sterylsulfatase may have functional implications in the placental uptake of circulating steroid sulfates.

Arylsulfatases↗

PubMed1989

Mannose 6-phosphate receptor in porcine thyroid follicle cells. Localization and possible implications for the intracellular transport of thyroglobulin.

Thyroglobulin has been shown to be phosphorylated and to carry the mannose 6-phosphate (M6P) signal in terminal position. In order to investigate whether the cation-independent mannose 6-phosphate receptor (CI-MPR) can possibly play a role in the transport of thyroglobulin the localization of the receptor was analyzed in thyroid follicle cells. The immunocytochemical observations showed that the CI-MPR is primarily located in elements of the endocytic pathway such as coated pits and endosomes. This localization of the CI-MPR in thyrocytes differs from the receptor sites in other cell types by the rare occurrence of the CI-MPR in cisternae of the Golgi complex. The observations are interpreted as an indication that the relatively small amount of receptor in the Golgi complex might be occupied primarily by lysosomal hydrolases. The CI-MPR in thyrocytes might, therefore, be unable to bind and to convey thyroglobulin efficiently. The receptor is, however, a binding site for thyroglobulin at the apical plasma membrane and may, therefore, be involved in the binding of thyroglobulin and its transfer from the follicle lumen to lysosomes.

Animals↗

PubMed1989

A colloidal gold labeling technique for the direct determination of the surface area of eukaryotic cells.

We have developed a colloidal gold labeling technique for the direct quantitation of the cell surface area. The method is based on coating the cell surface with [195Au] colloidal gold-protein complexes followed by morphometric determination of the labeling density (gold particles/micron2 cell surface) and radiometric determination of the total number of gold particles bound per cell. The ratio of both values directly gives the cell surface area. The accuracy of the method was shown using Staphylococcus aureus cells as a model system, where the cell surface area determined with our assay (4.0 microns2) corresponded well to the value calculated from the radius of the cells (3.6 microns2). In a more complex model system J-774 mouse macrophages were labeled with different amounts of [195Au] gold-protein complexes to show that the assay is independent of the degree of saturation of the cell surface binding sites. Both high (135 Au/microns2) and low (65 Au/microns2) labeling densities resulted in a surface area of about 1200 microns2. The technique finally was applied to L-929 fibroblasts to determine the increase of the cell surface area when the cells change from a spherical to a flat monolayer state. We found that the cell surface area increased 3-fold during the spreading process. The results show that the colloidal gold labeling technique allows the direct determination of the surface area of complex eukaryotic cells. The technique is suitable for the quantitation of changes in the surface architecture known to occur in different functional states of eukaryotic cells.

Animals↗

PubMed1989

[Possible decontamination of pancreatin with gamma rays and chemical agents].

Certain pre-conditions given, the microbial contamination of pancreatin in powder form can be reduced below international standard values. However, each of the methods whether physical or chemical, causes a variable loss of activity depending on the methods applied and the concentration of the decontaminating agent used. Therefore during preparation of this complex enzymatic compound, highly aseptic conditions must be kept in all stages of the process to avoid an increase of germal contamination. For a final toxicological assessment of the decontaminated products, further tests are required.

Chromatography, High Pressure Liquid↗

PubMed1988

Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes.

A monocytic cell line, termed Mono Mac, was established from peripheral blood of a patient with monoblastic leukemia. Two clones, designated Mono Mac I and Mono Mac 6, were isolated and both were assigned to the monocyte lineage on the basis of morphological, cytochemical and immunological criteria. Most importantly, the clones express NaF-sensitive non-specific-esterase, produce reactive oxygen and stain with MAb My4. Mono Mac 6, in addition, constitutively exhibits phagocytosis of antibody-coated erythrocytes in 80% of the cells and reacts with a panel of MAbs that are specific for mature monocytes, i.e., M42, LeuM3, 63D3, Mo2 and UCHMI. By contrast, the monoblastic cell lines U937 and THP-I are negative for all these markers. Only expression of My4 could be detected after differentiation induced by interferon-gamma (IFN-gamma). Similar treatment of Mono Mac I, however, resulted in staining with all the monocyte-specific MAbs mentioned above, while IFN-gamma treatment of Mono Mac 6 enhanced antigen expression. In addition, the cells showed an increased frequency of multinucleated cells with a rise from 4.8% to 21.9%. Mono Mac 6 appears to be the only one of the cell lines studied to constitutively express phenotypic and functional features of mature monocytes.

Cell Line↗

PubMed1988

Sulfation of thyroglobulin: a ubiquitous modification in vertebrates.

Mammalian thyroglobulin is released by thyroid follicle cells as a sulfated glycoprotein; the sulfate residues are mostly linked to tyrosine, but they are also attached to the high-mannose carbohydrate side-chains. To decide whether sulfation of thyroglobulin is confined to mammals, representatives of other vertebrate classes were analyzed for the presence of sulfated thyroglobulin: fish (trout), amphibians (clawed toad) and birds (chicken). Mini-organs were prepared from thyroid tissue and suspended in a 35SO4-(-)-containing culture medium. Light- and electron-microscope autoradiographs prepared from the mini-organs showed that thyroid follicle cells from all species examined incorporate 35SO4-(-) and synthesize a sulfated secretory product which accumulates in the follicle lumen. The Golgi complex was detected as the primary intracellular site of sulfate organification. The 35SO4-(-)-radiolabeled secretory product of all species was shown by polyacrylamide-gel-electrophoretic analyses to consist of thyroglobulin, identified by comparison with biosynthetically 125I-labeled thyroglobulin. The results indicate that the sulfation of thyroglobulin is a ubiquitous post-translational modification observed already in the thyroglobulin of lower vertebrates. Our observations suggest that sulfation of thyroglobulin was acquired in the early stages of thyroid evolution.

Animals↗

PubMed1988

Mini organ culture of thyroid tissue: a new technique for maintaining the structural and functional integrity of thyroid tissue in vitro.

We have developed a culture technique by which the normal configuration of follicles and most other structural characteristics of the thyroid gland are well preserved. Small tissue fragments (diameter 0.5 to 0.9 mm) from various mammalian species were prepared and kept in hydrophobic culture dishes to prevent their attachment. It was essential to raise the oxygen concentration in the incubator to 50% and the D-glucose concentration to 5.6 mM. Under these conditions, mini organs were formed by the outgrowth of epithelial cells from follicles located on the fragment surface and opened during tissue preparation. After 2 to 3 days, the mini organs were enclosed by a confluent and tight monolayer of follicle cells and [3H]thymidine incorporation returned to background levels. The structural integrity of follicles underneath the monolayer and in the center of the mini organs could be maintained for at least several weeks. Light microscope autoradiographs of mini organs incubated with iodide, sulfate, or phosphate revealed that the vectorial transport and the posttranslational modifications of thyroglobulin were maintained. An assay for the precise quantitation of biosynthetic activities in individual mini organs was developed. The observations indicate that thyroid-specific functions in mini organs can be preserved for prolonged periods of time.

Animals↗

PubMed1987

Thyroglobulin, the major and obligatory exportable protein of thyroid follicle cells, carries the lysosomal recognition marker mannose-6-phosphate.

Thyroglobulin (TG), the major exportable protein of thyroid follicle cells, is conveyed to lysosomes on a complex secretion, storage and recapture pathway by as yet unknown transport mechanisms. This report establishes that the dimeric porcine TG-molecule carries an average of six phosphate residues. Endoglycosidase digestion showed that two phosphate residues are bound to the high-mannose carbohydrate side chains (CHO), while two others are linked to the complex CHO. These four residues are also sensitive to alkaline phosphatase treatment, indicating their terminal linkage. Immunoprecipitation analyses showed that TG obtained from microsomal fractions is already phosphorylated. Most important, an enzymatic assay applied to hydrolysates of TG established that the two phosphate residues at the high mannose CHO are present as mannose-6-phosphate (M-6-P). Alkaline phosphatase treatment of biosynthetically radiophosphorylated CHO followed by hydrolysis and t.l.c. indicated that M-6-P is present at least in part in phosphomonoester linkage. Furthermore, porcine TG binds specifically to the M-6-P receptor of Chinese hamster ovary cells. It is concluded that the M-6-P residues of TG are exposed and able to operate as a ligand for the M-6-P receptor. It is unknown why the lysosomal recognition-marker M-6-P does not convey TG directly on an intracellular route to lysosomes. We propose that for the secretion of newly synthesized TG into the follicle lumen an additional export signal dominating over the M-6-P recognition-marker is required.

Animals↗

PubMed1987

Characterization of translocation contact sites involved in the import of mitochondrial proteins.

Import of proteins into the mitochondrial matrix requires translocation across two membranes. Translocational intermediates of mitochondrial proteins, which span the outer and inner membrane simultaneously and thus suggest that translocation occurs in one step, have recently been described (Schleyer, M., and W. Neupert, 1985, Cell, 43:339-350). In this study we present evidence that distinct membrane areas are involved in the translocation process. Mitochondria that had lost most of their outer membrane by digitonin treatment (mitoplasts) still had the ability to import proteins. Import depended on proteinaceous structures of the residual outer membrane and on a factor that is located between the outer and inner membranes and that could be extracted with detergent plus salt. Translocational intermediates, which had been preformed before fractionation, remained with the mitoplasts under conditions where most of the outer membrane was subsequently removed. Submitochondrial vesicles were isolated in which translocational intermediates were enriched. Immunocytochemical studies also suggested that the translocational intermediates are located in areas where outer and inner membranes are in close proximity. We conclude that the membrane-potential-dependent import of precursor proteins involves translocation contact sites where the two membranes are closely apposed and are linked in a stable manner.

Biological Transport↗

PubMed1987

Interactions between protein-gold complexes and cell surfaces: a method for precise quantitation.

We have developed a rapid and precise electron microscope technique for the quantitation of gold particles in suspension using latex microspheres as a reference (EM latex technique). This technique allowed us to determine the specific absorption of colloidal gold at its absorption maximum (520 nm) and the average number of ligands ([125I]IgG) bound to one gold particle. On the basis of these values important binding characteristics of protein-gold complexes to cell surfaces were analyzed in a model system consisting of Staphylococcus aureus with protein A on the cell wall as a specific binding site for IgG-Au. Our observations showed that the number of binding sites represented by one IgG-gold complex depended primarily on the particle size, with one 20-nm IgG-Au corresponding to 15 and one 6-nm IgG-Au to 2.5 binding sites. Hence, the efficiency of binding of IgG-Au complexes increased with decreasing gold particle size. Saturation of binding sites, however, was not achieved. The technique also made possible the determination of the affinity between IgG-Au complexes and the cell surface; this affinity can either be regarded as a characteristic of the ligand IgG or of the gold particle. We observed that the affinity of IgG decreased with the size of the gold particles to which IgG was bound, whereas the affinity of the entire gold particle increased with particle size. The EM latex technique for quantitation of gold particles extends the general use of protein-gold complexes to the quantitative characterization of their interaction with cell surface constituents.

Gold Colloid, Radioactive↗