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Biomedical subjects

J Roth

Publications and source records attributed to J Roth.

At least 541 records · Page 30Linked to original sources
PubMed1985

The ultrastructural labelling of keratin proteins in normal human colonic mucosa by a low temperature embedding, protein A-gold technique.

A low temperature embedding technique using protein A-gold as a marker was used to define the distribution of keratin proteins at the ultrastructural level in normal colonic epithelial cells. In both goblet and absorptive cells, keratin proteins were found at the base and lateral borders of the cell and inserting into desmosomes. There was a network of keratin filaments which surrounded cell organelles. In goblet cells, bundles of keratin filaments surrounded mucin droplets. In absorptive cells, a fine network of keratin filaments interdigitated with the terminal web. These studies with an immunochemical technique defined the keratin cytoskeleton at the ultrastructural level.

Colon↗
PubMed1985

Evolutionary origins of neuropeptides, hormones, and receptors: possible applications to immunology.

Immune function requires intercellular communication. The vocabulary includes messenger molecules closely linked to the immune system as well as more widely acting messengers such as hormones and neuroactive substances. To try to bring these together, we have used an evolutionary approach. Materials that resemble hormonal peptides and neuropeptides, previously thought to be restricted to multicellular animals, are present in protozoa, bacteria, and higher plants. There is also evidence for substances in microbes that bind hormones and other messengers, which resemble receptors of vertebrates. Therefore, we suggest that the molecules of intercellular communication probably arose much earlier in evolution than the endocrine, nervous, and immune systems. This insight provides new understanding of messenger systems in vertebrates, as applied to the immune system, as well as new insights into possible disease mechanisms, including those that involve autoimmunity.

Allergy and Immunology↗
PubMed1985

Functional asplenia in malignant mastocytosis.

A 47-yr-old man with malignant mastocytosis, a malignant neoplasia of mast cells, presented with anemia. Ultrasonography revealed an enlarged spleen. A liver-spleen scan using [99mTc]sulfur colloid failed to show any splenic uptake consistent with the diagnosis of functional asplenia. Functional asplenia related to this condition has not been reported previously. Since the spleen was heavily infiltrated with space occupying mast cells, a mass displacing effect along with impaired perfusion are discussed as underlying mechanism.

False Negative Reactions↗
PubMed1984

The role of antireceptor antibodies in stimulating phosphorylation of the insulin receptor.

Four polyclonal antisera directed against the insulin receptor were tested for their capability to activate the tyrosine-specific protein kinase associated with the receptor. All four antisera were shown to inhibit insulin binding to the receptor in cultured human lymphoblastoid cells and to stimulate lipogenesis in isolated rat adipocytes. Although two antisera (B-d, B-8) stimulated the activity of the tyrosine kinase of partially purified receptor preparations from rat liver, two other antisera (B-2 and B-10) failed to do so. This failure could not be explained by lack of antibody binding to receptor, by interference with the receptor as a substrate for the kinase, or by blocking of the enzyme's active site. We conclude that these two antireceptor antibodies bind to the receptor but fail to activate the kinase. The simplest interpretation of these observations is that activation of the tyrosine-specific protein kinase might not be an obligatory step in coupling insulin binding to insulin action. However, it is also possible that the mechanism by which polyclonal antireceptor antisera mimic insulin's bioactivity may differ from the mechanism of action of insulin itself.

Animals↗
PubMed1984

Immunohistochemical mapping of calcium-binding protein immunoreactivity in the rat central nervous system.

A complete mapping of immunoreactive sites for vitamin D-dependent calcium-binding protein (CaBP) was performed on serial sections from the rat central nervous system. CaBP immunoreactivity was found in the perikarya, dendrites and axons of some neurons from the limbic system, from many neurosecretory nuclei, from most sensory nuclei and from the cerebral and cerebellar cortex. In contrast, no CaBP antigenic sites were detectable in the motoneurons of the spinal cord and in those of the cranial nerve nuclei, nor in the neurons from the cerebellar nuclei. A quantitative evaluation revealed a great variability in the number of CaBP-immunoreactive neurons among different areas of the central nervous system. Positive cells represented less than 1% of the neurons in the frontal cortex, whereas 74% of the Purkinje cells from the cerebellar cortex showed immunoreactive staining for CaBP. In addition, 45% of the ependymal cells of the telencephalic ventricles were positive. These data show that CaBP is widely distributed in neurons and ependymal cells from the rat central nervous system although it is more concentrated in some specific areas.

Animals↗
PubMed1984

The insulin receptor of rat brain is coupled to tyrosine kinase activity.

Insulin receptors from rat brain were studied for receptor-associated tyrosine kinase activity. In solubilized, lectin-purified receptor preparations, insulin stimulated the phosphorylation of the beta subunit of its receptor as well as of exogenous substrates. Phosphoamino acid analysis of casein phosphorylated by these preparations revealed that 32P incorporation occurred predominantly on tyrosine residues. Receptor and casein phosphorylations were specific for insulin and analogues that also bind to the insulin receptor. The insulin dose response for phosphorylation of brain receptor resembled that reported for the purified insulin receptor from human placenta (Kasuga, M., Fujita-Yamaguchi, Y., Blithe, D.L., and Kahn, C.R. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2137-2141), suggesting similar insulin sensitivity and coupling of the brain receptor kinase. Four polyclonal antisera to the insulin receptor were able to bind and immunoprecipitate the brain receptor; however, only two antisera activated the receptor-associated kinase. Thus, the brain insulin receptor, like the well studied non-neural receptor, is coupled to tyrosine kinase activity, making regulation of cellular events by insulin in neural tissue possible.

Animals↗
PubMed1984

"Golden blot"--detection of polyclonal and monoclonal antibodies bound to antigens on nitrocellulose by protein A-gold complexes.

Protein A bound to particles of colloidal gold is widely used for cytochemical localization of antigens. A method for detecting immune complexes on "Western blots" with protein A-gold has been developed. It involved the following steps: (i) electrophoretic separation of proteins, (ii) transfer to nitrocellulose, (iii) incubation with antibodies, and (iv) staining with protein A-gold. The amount of antigen detectable was 10-50 ng. The sensitivity could be improved by subsequent photochemical silver staining (1-10 ng detected), and was then comparable to autoradiograms using iodinated protein A.

Animals↗
PubMed1984

Failure of bromocriptine to alter the qualitative characteristics of human growth hormone in acromegaly.

The role of bromocriptine as a therapeutic agent for acromegaly is uncertain. In the present study we have attempted to determine whether bromocriptine therapy causes qualitative changes in plasma human growth hormone (hGH) in acromegaly. When eight paired samples obtained before and during bromocriptine therapy were filtered over Sephadex G-100 there was no difference in the elution profiles. When the "little" hGH peak from each of the eight paired samples was pooled, lyophilized, and assayed in both radioreceptor assay (RRA) and radioimmunoassay (RIA), the RRA/RIA before treatment was not different than during treatment. When bromocriptine in pharmacologically significant concentrations was incubated with cultured human lymphocytes in vitro, there was no alteration in hGH binding properties. These results demonstrate directly that bromocriptine does not change the form or receptor-reactive properties of plasma hGH and, further, that the drug does not alter at least one form of human growth-hormone receptor.

Acromegaly↗
PubMed1984

Cytochemical localization of terminal N-acetyl-D-galactosamine residues in cellular compartments of intestinal goblet cells: implications for the topology of O-glycosylation.

The O-linked oligosaccharides of mucin-type glycoproteins contain N-acetyl-D-galactosamine (GalNAc) that is not found in N-linked glycoproteins. Because Helix pomatia lectin interacts with terminal GalNAc, we used this lectin, bound to particles of colloidal gold, to localize such sugar residues in subcellular compartments of intestinal goblet cells. When thin sections of low temperature Lowicryl K4M embedded duodenum or colon were incubated with Helix pomatia lectin-gold complexes, no labeling could be detected over the cisternal space of the nuclear envelope and the rough endoplasmic reticulum. A uniform labeling was observed over the first and several subsequent cis Golgi cisternae and over the last (duodenal goblet cells) or the two last (colonic goblet cells) trans Golgi cisternae as well as forming and mature mucin droplets. However, essentially no labeling was detected over several cisternae in the central (medial) region of the Golgi apparatus. The results strongly suggest that core O-glycosylation takes place in cis Golgi cisternae but not in the rough endoplasmic reticulum. The heterogenous labeling for GalNAc residues in the Golgi apparatus is taken as evidence that termination of certain O-oligosaccharide chains by GalNAc occurs in trans Golgi cisternae.

Acetylgalactosamine↗
PubMed1984

Unique features of the insulin receptor in rat brain.

We examined the structure of the affinity-labeled insulin receptors in rat brain, rat liver, and human IM-9 lymphocytes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In gels run under reducing conditions, the alpha-subunit of the insulin receptor in brain had an apparent Mr of 127,000 distinctly lower than that seen in both rat liver and human lymphocytes (apparent Mr = 136,000). Exposure to neuraminidase increased the electrophoretic mobility of the liver receptor, but had no effect on the insulin receptor in brain. The carbohydrate moieties of the insulin receptors in rat brain and liver were further examined by chromatography on wheat-germ agglutinin agarose. The receptors in both tissues adsorbed to the wheat-germ agglutinin; elution with 0.3 M N-acetyl glucosamine resulted in slightly better recovery of the brain than of the liver receptor. Exposure to neuraminidase virtually abolished the interaction of the liver receptor with the lectin, whereas adsorption of the brain receptor was unaffected by neuraminidase. These results indicate that the insulin receptor in brain is distinguished from those in peripheral tissues by structural alterations, including changes in the carbohydrate moiety of the receptor. Such alterations contrast sharply with the previously observed similarities in insulin binding properties between insulin receptors in brain and other tissues. The implications of such structural alterations for the program of insulin action expressed by the receptors in brain remain to be explored.

Affinity Labels↗
PubMed1984

Insulin receptor degradation is accelerated in cultured lymphocytes from patients with genetic syndromes of extreme insulin resistance.

Previous studies of the insulin receptor in disease states have utilized primarily techniques of equilibrium binding and, to a limited extent structural, analysis. Though techniques have been developed to study receptor degradation in normal cells, they have not been applied to disease states. In the present study we have examined insulin receptor degradation rate in B lymphocytes that were obtained from peripheral blood of normal subjects and patients with several syndromes of extreme insulin resistance. B lymphocytes were established in culture from each patient's peripheral cells by transformation with Epstein-Barr virus. The insulin receptors were surface labeled using Na125I/lactoperoxidase and the cells were returned to incubate in growth media. After varying periods of incubation, aliquots of cells were solubilized and the cell content of labeled receptor subunits were measured by immunoprecipitation with anti-receptor antibodies and NaDodSO4/polyacrylamide gel electrophoresis. The fall in 125I-insulin receptor content approximated a single exponential and was quantitated as receptor subunit half-life (t1/2). In cell lines from four patients in whom the number of insulin receptors was reduced by greater than 90%, the rate of receptor loss was greater than normal (t1/2 equals 3.8 +/- 0.9 h vs. 6.5 +/- 1.2 h; mean +/- SD, P less than 0.01). However, a similar acceleration in receptor degradation was seen in cells from five patients with extreme insulin resistance but low-normal insulin receptor concentration (t1/2 equals 4.4 +/- 0.9 h). This group included cells from one patient with a qualitatively abnormal receptor. Thus, all the patients with genetic syndromes of insulin resistance had accelerated receptor degradation, regardless of their receptor concentration. By contrast, insulin receptors on cultured lymphocytes that were obtained from patients with extreme insulin resistance secondary to autoantibodies to the insulin receptor had normal receptor degradation (t1/2 equals 6.1 +/- 1.9 h). We conclude that (a) accelerated insulin receptor degradation is an additional feature of cells from patients with genetic forms of insulin resistance; (b) that accelerated insulin receptor degradation may explain the low-normal receptor concentrations that were seen in some patients with extreme insulin resistance; and (c) that accelerated degradation does not explain the decreased receptor concentration in patients with very low insulin receptor binding and, therefore, by inference, a defect in receptor synthesis must be present in this subgroup.

Adolescent↗
PubMed1984

Applications of immunocolloids in light microscopy. III. Demonstration of antigenic and lectin-binding sites in semithin resin sections.

Previous studies have demonstrated that antigens or lectin-binding sites can be localized in sections from paraffin-embedded tissues with protein A or lectins bound to colloidal gold or colloidal silver (Roth J: J Histochem Cytochem 30:691, 1982 and 31:547, 1983). In the present study the protein A-gold technique and lectin-gold complexes have been applied to semithin sections (0.5-1.5 micron) of Epon- or low temperature Lowicryl K4M-embedded rat pancreas, kidney and submandibular gland. The results show that an increase in resolution and, therefore, in amount of information can be obtained. The optimal mode of imaging was determined on sections without counterstaining. Bright-field illumination gives the maximum information about the staining signal, while phase-contrast and Nomarski differential interference contrast give predominantly structural and, to a lesser extent, staining information. Polarization epi- and transillumination microscopy is inferior in all aspects. The application of a battery of lectin-gold complexes to rat submandibular gland revealed a specific staining pattern for each lectin in acinar and excretory duct cells.

Animals↗
PubMed1984

Light and electron microscopic demonstration of sialic acid residues with the lectin from Limax flavus: a cytochemical affinity technique with the use of fetuin-gold complexes.

The development of a cytochemical affinity technique for the demonstration of sialic acid residues by light and electron microscopy is reported. The lectin from the slug Limax flavus, with its narrow specificity for N-acetyl- and N-glycolylneuraminic acid, was applied to tissue sections. Subsequently fetuin-gold complexes were used to visualize the tissue-bound lectin. Different cytochemical controls, including sugar inhibition tests, neuraminidase digestion, the use of fetuin-gold complexes alone, or acid hydrolysis of sections, proved the specificity of the technique. Postembedding staining was performed on frozen, paraffin, or semithin resin sections for light microscopy and on thin sections from low temperature Lowicryl K4M-embedded material for electron microscopy. The distribution of sialic acid residues in rat pancreas, liver, and colonic mucosa was investigated.

Animals↗
PubMed1984

Early development and tissue-specific patterns of insulin binding in chick embryo.

We studied the development of insulin binding to its receptor in chick embryos from days 2-18 of the 21-day incubation period. Using partially purified membrane preparations we found that insulin receptors on both brain and liver of day 18 embryos were typical insulin receptors by multiple criteria. Specific insulin binding to preparations of whole embryos was not detected on day 2, but was present by day 3. Insulin receptors were present on both heads and bodies of embryos by day 4. Binding to liver and brains increased with development between days 8 and 18; however, the patterns of increase in the two tissues were quite distinct. In liver, binding was low on day 8, but increased markedly by day 14 (P less than 0.01). Increases after day 14 were less impressive. In contrast, binding to brain preparations was relatively well established by day 8 and did not change significantly between days 8 and 14. An abrupt increase occurred between days 14 and 16 (P less than 0.01), and a second increase took place between days 16 and 18. We have previously shown that insulin is present in chick embryos before known pancreatic development. The current demonstration that insulin receptors are also present early in embryogenesis makes it possible that insulin may influence the embryo at early stages. In addition, the different patterns of increases in insulin binding in liver and brain during mid- to late development suggest complex and tissue-specific mechanisms of regulation.

Animals↗
PubMed1984

Downregulation occurs normally in cultured Epstein-Barr virus-transformed lymphocytes from patients with extreme insulin resistance. Discrepancy between downregulation in vivo and in vitro.

Levels of fasting plasma insulin are generally inversely correlated with 125I-insulin binding to circulating blood cells. In disease states associated with hyperinsulinemia (e.g., obesity and non-insulin-dependent diabetes mellitus), 125I-insulin binding is usually low. In contrast, 125I-insulin binding to circulating cells may be normal in patients with certain forms of extreme insulin resistance despite marked hyperinsulinemia. To explain this paradox, it has been proposed that postbinding defects in insulin action may give rise to defects in downregulation. We have employed cultured Epstein-Barr virus (EBV)-transformed lymphocytes from eight patients with extreme insulin resistance to address the question of whether there is a defect in the downregulation process in vitro. In this cell type, insulin leads to a decrease in the number of insulin receptors on the cell surface by accelerating the rate of degradation of insulin receptors. We could not detect any abnormality in in vitro down-regulation with cultured EBV-transformed lymphocytes from insulin-resistant patients. The apparent discrepancy between the in vivo and in vitro studies raises the possibility that some factor in the patient's internal milieu may prevent insulin-induced downregulation. An alternative possible explanation might be that the mechanism of downregulation in vitro differs from the mechanism whereby receptor number is regulated in vivo in insulin's target cells.

Adolescent↗