Syndromes associated with inappropriate hormone synthesis by tumors: an evolutionary interpretation.
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Animals↗
Biomedical subjects
J Roth
Publications and source records attributed to J Roth.
"Thiocyanate gold": small (2-3 nm) colloidal gold for affinity cytochemical labeling in electron microscopy.
Reduction of HAuCl4 by NaSCN or KSCN produces colloidal gold particles of 2.6 nm in diameter and homogeneous in size (coefficient of variation approximately 15%). The AuSCN sol forms protein-gold complexes. The amount of protein required to form an AuSCN-protein complex is best determined in the electron microscope, where serial dilutions of protein with gold sol are inspected for the presence of aggregates. By immuno-electron microscopy SCN-gold complexed to protein A is active and visible as is shown by revealing alpha-amylase in rat pancreatic acinar cells.
Animals↗
Lyophilization of protein-gold complexes.
Conditions for dialysis, freezing and lyophilization of protein-gold complexes were established. Lyophilized complexes formed by colloidal gold with staphylococcal protein A, with Helix pomatia lectin and with horseradish peroxidase retained their original staining properties when reconstituted after several months of storage.
Animals↗
Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation.
Sialyltransferase (Gal beta 1,4GlcNAc alpha 2,6 sialyltransferase) was localized by immunoelectron microscopy in rat liver hepatocytes using affinity-purified antibodies. Immunoreactivity for sialyltransferase was found in the Golgi apparatus, where it was restricted to an interconnected system consisting of the trans-cisternae and the trans-tubular network. This region of the Golgi apparatus exhibited both TPPase and CMPase activity and was the intracellular site where sialic acid residues bound to glycoprotein were detected using the Limax flavus lectin. Sialyltransferase and sialic acid residues were not detected in medial and cis-cisternae of the Golgi apparatus. These findings suggest that in rat hepatocytes sialylation of N-linked glycoproteins occurs in the complex formed by the trans-cisternae and the trans-tubular network of Golgi apparatus.
Animals↗
What's new in light and electron microscopic immunocytochemistry? Application of the protein A-gold technique to routinely processed tissue.
The protein A-gold technique, a simple and reliable two-step postembedding immunocytochemical method, allows the light and electron microscopic detection of antigens in routinely fixed and embedded tissue. At the light microscopic level a permanent, nonfading stain is obtained. High resolution studies on intracellular antigens can be performed since the particulate nature of the colloidal gold permits accurate determination of the labeled cellular structures. The applicability of the protein A-gold technique for the localization of various peptide and protein antigens by light and electron microscopy is demonstrated.
Adenoma↗
Salmon calcitonin-like immunoactivity in extracts of Tetrahymena pyriformis.
Acid extracts of Tetrahymena pyriformis, a ciliated protozoan grown in defined medium revealed the presence of materials with salmon-type calcitonin immunoactivity. These findings add support to earlier reports of the presence of materials closely resembling vertebrate peptide hormones in unicellular microbes.
Animals↗
Transport to cell surface of intestinal sucrase-isomaltase is blocked in the Golgi apparatus in a patient with congenital sucrase-isomaltase deficiency.
A case of congenital sucrase-isomaltase deficiency in man was investigated. An intestinal biopsy sample from a 5-year-old girl lacked sucrase but possessed low residual isomaltase activity. Immunoelectron microscopy with monoclonal antibodies to sucrase-isomaltase in biopsy samples from healthy subjects revealed that sucrase-isomaltase was confined predominantly to the microvillus membrane of enterocytes and there was minimal labeling of the Golgi apparatus. In the patient immunoreactive sucrase-isomaltase was found almost exclusively in about three trans-Golgi cisternae and associated vesicular structures, while no specific labeling was associated with the microvillus membrane. Immunoprecipitation experiments with iodinated mucosal homogenates and a mixture of four monoclonal antibodies to sucrase-isomaltase revealed absence of enzyme subunits in the patients but presence of a Mr 210,000 protein that was also expressed in normal control biopsy specimens. This protein presumably is the high-mannose precursor of sucrase-isomaltase. Additional proteins of Mr 160,000-200,000 found in the patient but not in normal subjects might correspond to the crossreacting material found in the Golgi apparatus of the patient. Overall, the findings suggest that in the patient sucrase-isomaltase is synthesized and transported to the Golgi apparatus, where further transport is interrupted. The data imply that signals in sucrase-isomaltase that mediate its transfer from the endoplasmic reticulum to the Golgi apparatus differ from those mediating its transport from the Golgi apparatus to the cell surface.
Antibodies, Monoclonal↗
Localization of sialic acid in kidney glomeruli: regionalization in the podocyte plasma membrane and loss in experimental nephrosis.
Sialic acid residues were localized by electron microscopy in renal glomeruli of normal and puromycin-treated rats with a cytochemical technique that utilized the Limax flavus lectin. In Lowicryl K4M thin sections from normal rats, sialic acid residues were found along the plasma membrane of the various glomerular cell types and in the glomerular basement membrane as well as the mesangial matrix. In NaDodSO4/PAGE, sialic acid residues of normal glomeruli were mainly confined to a 140-kDa protein previously identified as podocalyxin. The distribution of sialic acid residues in the podocyte plasma membrane was found to be remarkably regionalized. Based on the differential labeling intensity, three plasma membrane domains could be defined: the foot process base, the foot process region above the slit diaphragm, and the body of podocytes. Cytochemical and biochemical analysis of glomeruli from puromycin-treated rats showed a loss of sialic acid residues from glomerular sialoglycoconjugates indicating a perturbated glycosylation.
Animals↗
Immunocytochemical demonstration of ecto-galactosyltransferase in absorptive intestinal cells.
Galactosyltransferase immunoreactive sites were localized in human duodenal enterocytes by the protein A-gold technique on thin sections from low temperature Lowicryl K4M embedded biopsy specimens. Antigenic sites detected with affinity-purified, monospecific antibodies were found at the plasma membrane of absorptive enterocytes with the most intense labeling appearing along the brush border membrane. The lateral plasma membrane exhibited a lower degree of labeling at the level of the junctional complexes but the membrane interdigitations were intensely labeled. The labeling intensity decreased progressively towards the basal part of the enterocytes and reached the lowest degree along the basal plasma membrane. Quantitative evaluation of the distribution of gold-particle label proved its preferential orientation to the outer surface of the plasma membrane. In addition to this membrane-associated labeling, the glycocalyx extending from the microvillus tips was heavily labeled. Occasionally, cells without plasma membrane labeling were found adjacent to positive cells. The demonstration of ecto-galactosyltransferase on membranes other than Golgi membranes precludes its general use as a marker for Golgi membrane fractions. The possible function of galactosyltransferase on a luminal plasma membrane is unclear at present, but a role in adhesion appears possible on the basolateral plasma membrane.
Antibodies↗
Ultrastructural localization of keratin proteins in human skin using low-temperature embedding and the protein A-gold technique.
Human skin was embedded in Lowicryl K4M and keratin proteins were localized by incubation with antikeratin antisera, followed by protein A-gold. The antikeratin antisera labeled all intermediate filament (tonofilament) structures in all layers of the epidermis. The association of keratin filaments with hemidesmosomes, desmosomes, and keratohyaline granules was clearly visualized. Desmosomes and keratohyaline granules were not labeled by the antikeratin antisera. No nonfilamentous structures were labeled. The technique described is suitable for studying the distribution of keratin filaments in normal and diseased tissue.
Acrylic Resins↗
Theoretical calculation of layer geometry in rotary shadowed models of membrane-associated particles.
The geometry of the evaporated metal layer on models of membrane-associated particles has been calculated theoretically and the respective density profiles have been estimated. The models were a hollow cylinder, a hemisphere and a rotary ellipsoid substituting the freeze-fracture appearance of gap junction connexons and globular integral membrane proteins respectively. These calculations may be used as a help for choosing an optimal compromise between shadowing angle and layer thickness. They furthermore provide a basis for assaying the extent of shadowing artefacts such as decoration and lead to a more accurate interpretation of rotary shadowed structures.
Animals↗
Insulin-related material in microbes: similarities and differences from mammalian insulins.
We have reported that extracts of Escherichia coli and Tetrahymena grown in synthetic media contained material that reacted specifically in the immunoassay and bioassay for insulin. One additional strain of Tetrahymena and four of E. coli yielded amounts of material similar to those reported previously. In addition to their behavior on Sephadex G-50, the immunoactive insulin-related materials from the microbial sources behaved like authentic vertebrate insulins in their ability to be adsorbed to and eluted from disposable octadecasilylsilica cartridges, DEAE-Sephadex, DEAE-cellulose, and one system of high-pressure liquid chromatography (HPLC). As with less purified microbial material, the "insulin" that had been purified on DEAE and HPLC, when tested for its bioactivity, had an immunoactivity:bioactivity ratio of approximately unity and the bioactivity was largely neutralized by anti-insulin antibody. Because the material from the microbes was so similar to authentic insulins, studies were undertaken to demonstrate that inadvertent contamination with vertebrate insulins was highly unlikely. Blanks carried through the entire procedure were always negative. Tetrahymena grown and extracted in another laboratory gave the same results. Tetrahymena that had been grown but then allowed to stand in the fermenter under adverse conditions and then carried through the entire procedure were devoid of insulin. Tetrahymena that were homogenized and subjected to acid hydrolysis were devoid of insulin. Further substantiation that exogenous contamination was highly unlikely was provided by two other types of experiments. In one of these, it was shown that the subcellular distribution of exogenously added porcine insulin or porcine 125I-labeled insulin was different from the distribution of endogenous insulin. In the second type of experiment, it was shown that during the log phase of growth of Tetrahymena or of E. coli the insulin content of the system increased multifold in a fashion that might be expected for living organisms but quite unexpected for exogenous contamination. (Interestingly, the insulin content of the E. coli medium far exceeded that which might be contributed by death of cells, estimated by the content in the medium of an intracellular enzyme.) When E. coli was grown and processed in four other laboratories having no contact with our own, similar levels of insulin-related material were recovered.
Animals↗
Lectin-gold cytochemistry reveals intercalated cell heterogeneity along rat kidney collecting ducts.
The lectin-gold technique was used to detect Helix pomatia and Dolichos biflorus lectin binding sites directly on semithin and thin sections of rat kidney collecting ducts. Intercalated cell apical plasma membranes and the membranes of apical cytoplasmic vesicles were heavily labeled in the cortex and outer stripe of the outer medulla but were negative or very weakly labeled in the inner stripe and inner medulla. In contrast, clear cell apical membranes were labeled along the entire length of the collecting duct. Double labeling of semithin cryostat sections with a specific antibody and lectin-gold complexes was used to demonstrate that the intercalated cells in all regions studied contained carbonic anhydrase, even though the lectin binding differed. These results indicate that, in terms of their glycocalyx composition, intercalated cells represent a heterogeneous population in different regions of the collecting duct.
Animals↗
Applications of immunocolloids in light microscopy. IV. Use of photochemical silver staining in a simple and efficient double-staining technique.
We report the development of a new light-microscopic double-staining technique using colloidal gold as sole marker. The contrasting color to the red of colloidal gold is achieved by the application of photochemical silver reaction. The silver reaction, which is principally performed at the end of the first staining sequence, converts the red color of a gold-labeled reagent into black. This contrasts clearly with the red coloration that results from the second incubation sequence without silver reaction. For antigen double staining, the same protein A-gold complex can be used to provide the black and the red color, thus rendering the technique very economical. Alternatively, combination of protein A-gold immunolocalization and lectin-gold staining is possible, as is combined lectin-gold staining.
Animals↗
Immuno-ultrastructural localization of involucrin in squamous epithelium and cultured keratinocytes.
Involucrin immunoreactivity was localized ultrastructurally with protein A--gold in epidermis and cultured keratinocytes embedded in Lowicryl K4M. In the skin, immunoreactivity was found predominantly in cells of the granular layer and inner stratum corneum. The label was associated primarily with amorphous cytoplasmic material and especially keratohyaline granules. Some labeling was observed at the cell periphery, but little with keratin filaments. Tissue samples examined without aldehyde fixation showed relatively greater labeling in the outer stratum corneum than fixed tissue. In cultured cells, the labeling was also associated primarily with cytoplasmic granular material and to a lesser extent with the cell periphery. Upon treatment with the ionophore X537A, keratin filaments were found in aggregated arrays and the plasma membranes became convoluted. That involucrin immunoreactivity persisted in the cytoplasm in cultured cells and in vivo after cross-linking occurs could account for considerable isopeptide bonding detected in epidermal keratin fractions and indicates that not all the involucrin participates in envelope formation.
Cells, Cultured↗
Ontogeny of receptors for insulin-like peptides in chick embryo tissues: early dominance of insulin-like growth factor over insulin receptors in brain.
Recently, we confirmed early data showing deleterious effects of exogenous insulin on chick embryos at 2 days of development, although insulin receptors were not clearly demonstrable until days 3-4. Now we report that insulin-like growth factor (IGF) receptors are present in whole embryos on day 2. The developmental patterns of [125I]IGF-I and [125I]IGF-II binding to brain were similar, and IGF-I showed approximately a 2-fold higher binding than IGF-II; there was a sharp increase from days 3 to 6, and a subsequent gradual fall during the second and third weeks of ontogeny. Competitive binding experiments with unlabeled analogs suggested that both labeled IGFs were binding to type I IGF receptors, and insulin interacted with them. The temperature and pH dependence were relatively higher than those for some other known IGF receptors. We have previously reported that [125I]insulin binding to brain is barely detectable on day 3 and shows a progressive rise throughout the rest of embryonic life. The pattern of IGF and insulin receptors appears to be organ specific, since nonneural tissues such as heart, liver, and limb buds showed different binding profiles in ontogeny. We conclude from these data that IGF receptors develop in chick embryo brain before insulin receptors and probably can mediate effects of IGF and insulin at early stages of embryogenesis.
Animals↗
Insulin-stimulated receptor phosphorylation appears normal in cultured Epstein-Barr virus-transformed lymphocyte cell lines derived from patients with extreme insulin resistance.
Insulin-stimulated phosphorylation of the insulin receptor was studied in cultured B-lymphocytes transformed by Epstein-Barr virus. In studies with cell lines derived from six normal subjects, insulin (10(-7) M) caused an average increase of approximately 200% in 32P incorporation into the 95K subunit of the insulin receptor. Phosphorylation was rapid (detectable within 1-2 min) and reached a maximum level by 15 min. Dose-response curves for receptor occupancy and phosphorylation were nearly superimposable, indicating few or absent spare receptors for this response to insulin. These data suggest that insulin receptor phosphorylation is an early response to insulin in cultured lymphocytes transformed with Epstein-Barr virus. We studied insulin receptor phosphorylation in cell lines derived from nine patients with clinical syndromes associated with extreme insulin resistance, all of whom had normal [125I] insulin binding. While the magnitude of insulin's stimulation varied widely among the individual cell lines, no significant differences were found between cell lines from normal subjects and those from patients with extreme insulin resistance.
Cell Line↗
Insulin antibodies retard and insulin accelerates growth and differentiation in early embryos.
The physiologic function of insulin in early embryonic life is unknown. We have shown that insulin is present in unfertilized eggs and in chick embryos at 2-3 days of development, even before the emergence of the endocrine pancreas. To define insulin's role, we exposed 2-day-old chick embryos to anti-insulin antibodies and followed their development up to day 5. Antibody-treated embryos had a higher rate of growth retardation and death by days 3-5 of embryogenesis, compared with controls. Among the survivors, biochemical maturation was delayed at days 4 and 5; weight, protein, total creatine kinase activity, and creatine kinase-MB were decreased in antibody-treated embryos. By contrast, insulin (50 ng/embryo) administered to 2-day-old embryos yielded nearly symmetrical stimulatory results. These findings suggest that endogenous insulin plays a probable physiologic role regulating growth and differentiation in early embryos. In addition, the findings provide some clues to a possible function for insulin produced outside the organism's own beta cells.
Animals↗