Search PubMed⌕ Search

Biomedical subjects

D LeRoith

Publications and source records attributed to D LeRoith.

At least 253 records · Page 14Linked to original sources
PubMed1986

Extracts of protozoa contain materials that react specifically in the immunoassay for guinea pig insulin.

Extracts of protozoa contain materials that resemble guinea pig insulin, which is noted for its unusual structure and properties. The protozoan derived materials react in the radioimmunoassay for guinea pig insulin; some but not all of these immunoreactive materials migrate on gel filtration in the position of authentic guinea pig insulin. Experiments were done to exclude artifacts in the assay as well as inadvertent contamination by guinea pig insulin. By immunological methods, we segregated the guinea pig type immunoactivity from that which has rat/pork type immunoactivity. These findings extend our studies of extracts of guinea pig tissues which also have these two types of insulin immunoactivities.

Animals↗
PubMed1986

Insulin receptors from guinea pig liver and brain: structural and functional studies.

We studied the structural and functional characteristics of insulin receptors from guinea pig liver and brain. Binding to crude membrane preparations of liver and brain was time, temperature, and pH dependent. Maximal specific binding to liver crude membrane preparations was 16.4 +/- 0.5%, and that to brain crude membrane preparations was 10.4 +/- 1.8%. Specificity studies demonstrated typical affinities for insulin receptors with chicken insulin greater than porcine insulin greater than human proinsulin greater than desoctapeptide insulin in both liver and brain. Antiinsulin receptor antiserum inhibited binding of [125I]insulin to both liver and brain crude membrane preparations. Electrophoresis performed under reducing conditions after affinity cross-linking of liver and brain insulin receptors with [125I]insulin revealed labeled proteins (alpha-subunit) with apparent mol wt of 136,000 in liver and 121,000 in brain. Treatment of liver and brain receptors with both endoglycosidase H and endoglycosidase F increased the electrophoretic mobility of the alpha-subunit. [125I]Insulin cross-linked receptors from both liver and brain adsorbed to and eluted from wheat germ agglutinin columns in a similar manner, as demonstrated by binding and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In solubilized lectin-purified receptor preparations from liver and brain, insulin stimulated the phosphorylation of the beta-subunit and exogenous substrates. When the delta-kinase activity, as measured by exogenous substrate phosphorylation, of the brain and liver preparations was normalized to the maximal bound to free ratio of the preparations, the delta-kinase activity was about 4-fold greater in brain than in liver. These studies suggest that the differences between brain and liver insulin receptor alpha-subunits previously demonstrated in rats are also present in guinea pigs.

Acetylglucosaminidase↗
PubMed1986

Development of brain insulin receptors: structural and functional studies of insulin receptors from whole brain and primary cell cultures.

We studied the structural and functional characteristics of insulin receptors from rat brain and liver from late gestation through adulthood as well as from cultured neuronal and glial cells from neonatal rats. Specific insulin binding was present on membrane preparations from brain and liver at all stages of development studied, with maximal binding in neonates greater than 19-day-old fetuses greater than adults for both brain and liver. Maximal specific binding to cultured neuronal and glial cell membranes was similar (6.2% vs. 7.1%, respectively). [125I]Iodoinsulin cross-linking to the insulin receptor demonstrated that the mol wt (Mr) of the brain alpha-subunit was less than that of the liver alpha-subunit at all stages. [125I]Iodoinsulin cross-linking also demonstrated that the glial cell alpha-subunit (Mr, 130,000) migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to a position intermediate between the liver (Mr, 135,000) and brain (Mr, 119,000), whereas the neuronal cell alpha-subunit (Mr, 118,000) comigrated with the brain alpha-subunit. In solubilized lectin-purified preparations from brain and liver during development as well as from neuronal and glial cells, insulin stimulated phosphorylation of the beta-subunit. The Mr of the brain beta-subunit, as determined by migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was less than that of the liver beta-subunit. The neuronal cell beta-subunit comigrated with the brain beta-subunit while the glial cell beta-subunit migrated to a position intermediate between the brain and liver beta-subunit. Solubilized lectin-purified preparations from all tissues demonstrated insulin-stimulable phosphorylation of exogenous substrates. From these studies we conclude that 1) functional insulin receptors are present in the brain during development in the rat; and 2) the structural differences demonstrated between neuronal and glial cell and between brain and nonneuronal insulin receptors taken together with previously demonstrated functional differences of the insulin receptor on these tissues suggest a unique function for insulin receptors on neuronal tissues.

Age Factors↗
PubMed1985

Bacillus subtilis contains multiple forms of somatostatin-like material.

Extracts of B. subtilis contain somatostatin-like immunoactivity (1-20 pg per g wet weight cells). Two major forms were detected, one with reactivity in both N- and C-terminal immunoassays similar to somatostatin-28 and a second form reactive only in the C-terminal specific immunoassay similar to somatostatin-14. Both forms were active in a bioassay and the bioactivity was neutralized in the presence of antibody to the central, biologically active part of somatostatin-14. Preconditioned medium contained no detectable somatostatin whereas conditioned medium had 80-380 pg per liter.

Animals↗
PubMed1985

Evidence for multiple molecular weight forms of somatostatin-like material in Escherichia coli.

Extracts of Escherichia coli grown in defined medium contain somatostatin-related material (1-10 pg/g wet weight of cells). Preconditioned medium had no immunoactive somatostatin whereas, conditioned medium had 110-150 pg/l. Following purification of the extracted material on Sep-pak C18, Bio-Gel P-6 and HPLC, multiple molecular weight forms of somatostatin- (SRIF-) related material were identified. The material in one peak reacted in both the N-terminal and C-terminal SRIF immunoassay and coeluted on HPLC with SRIF-28, whereas that in a second peak eluted near SRIF-14 and was reactive only in the C-terminal SRIF assay. The two peaks are thus similar to SRIF-28 and SRIF-14 of vertebrates. These findings add support to the suggestion that vertebrate-type peptide hormones and neuropeptides have early evolutionary origins.

Chromatography, Gel↗
PubMed1985

Two distinct insulin-related molecules in the guinea pig: immunological and biochemical characterization of insulin-like immunoactivity from extrapancreatic tissues of the guinea pig.

In this study we extracted guinea pig brain and testis with; the extract was adsorbed to and eluted from cartridges (the Sep-Pak C18 procedure). We found this procedure superior for recovering crystalline insulin added to buffers or tissues, and for recovering endogenous insulin from plasma, but inferior for recovery of insulin from tissues. However, we did find 'rat/pork' type-insulin in guinea pig brain and testis (5-50 pg/g wet weight tissue). Our results with the Sep-Pak C18 procedures were reproduced by four other laboratories (who found 4-60 pg/g wet weight of tissue) and similar findings were also obtained by an independent investigator. Thus, we conclude that extrapancreatic tissues of guinea pigs have a second type of insulin-related material that is more typical of other mammalian insulins, but that the amount recovered is dependent upon the extraction procedure utilized.

Animals↗
PubMed1985

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↗
PubMed1985

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↗
PubMed1985

Somatostatin-like material is present in flowering plants.

Extracts of spinach contain somatostatin (SRIF)-related material (6-80 pg/g wet wt). The SRIF-related material, when purified on HPLC, was recovered as two major mol wt forms; one that eluted with a retention time similar to that of synthetic SRIF-28 and reacted in both N- and C-terminal-specific immunoassays, and a second peak that eluted with a retention time similar to that of SRIF-14 and reacted only in the C-terminal immunoassays. The purified material was active in a sensitive bioassay, and the bioactivity was neutralized in the presence of anti-SRIF antiserum. Since we have previously described the presence of similar material in bacteria, we also tested extracts of the flowering plant Lemna gibba G3, which was grown under sterile conditions. The Lemna extracts also had SRIF-related material (3.0 pg/g wet wt). Since plants are probably derived evolutionarily from unicellular organisms, the presence of SRIF-like material in higher plants gives support for the hypothesis that vertebrate-type peptide hormones have early evolutionary origins.

Animals↗
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↗
PubMed1982

Betazole-induced GIP secretion is not mediated by gastric HCl.

Betazole, a pyrazole analogue of histamine, as well as pentagastrin and HCl stimulate GIP secretion. We have asked the question as to whether betazole acts directly or via the production of HCl. Eight normal subjects and 4 patients with achlorhydria secondary to pernicious anemia were given betazole (0.5 mg/kg) by IM injection. Another six normal subjects were also given betazole but this was preceded by 200 mgs. of the H2 receptor blocker cimetidine given IV 60 mins. previously and a slow infusion of 200 mg. cimetidine given over the next 4 hr. Our results have shown that the GIP response to betazole is maintained in achlorhydric subjects as well as during H2 blockade. The results suggest that betazole and therefore histamine may stimulate GIP directly and not necessarily via the mediation of HCl.

Achlorhydria↗