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Biomedical subjects

J Roth

Publications and source records attributed to J Roth.

At least 613 records · Page 34Linked to original sources
PubMed1981

Enzymatic properties of the purified putA protein from Salmonella typhimurium.

In the previous paper (Menzel, R., and Roth, J. (1981) J. Biol. Chem. 256, 9755-9761) we have described the purification of a protein, the putA gene product, which has both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities. In this paper we demonstrate that these enzyme activities are distinct with respect to a number of characteristics. The oxidase activity proceeds by a ping-pong mechanism involving the reduction of an enzyme-bound flavin. The dehydrogenase activity utilizes an ordered reaction mechanism.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1981

Qualitative abnormalities in insulin binding in a patient with extreme insulin resistance: decreased sensitivity to alterations in temperature and pH.

Cultured lymphocytes transformed by Epstein--Barr virus were employed to study insulin receptors from a patient with extreme insulin resistance associated with the syndrome of leprechaunism. With cultured lymphocytes from normal subjects, insulin binding to its receptor is exquisitely sensitive to changes in temperature and pH. In cells from normal subjects, insulin binding was increased by approximately 250% as the temperature was decreased from 37 degrees C to 12 degrees C. In contrast, with cells from the leprechaun, insulin binding was only approximately 30% higher at 12 degrees C than at 37 degrees C. Similarly, insulin binding to cells from the leprechaun was markedly less sensitive to changes in pH, as compared to cells from normal subjects. Binding studies suggested that the number of insulin receptors per cell was within the normal range in this patient. Despite the unusual characteristics of insulin binding in cells from this insulin-resistant patient, the receptors were typical in at least two respects: (i) binding was inhibited normally by antibodies to the receptor; and (ii) the specificity for insulin analogs was normal (chicken insulin greater than porcine insulin much greater than guinea pig insulin greater than porcine proinsulin). This patient has an inborn error affecting insulin receptor function. The receptor's binding function was abnormal in having decreased sensitivity to alterations in temperature and pH. However, the level of insulin binding to cells from the leprechaun was within normal limits. Consequently, the hormonal resistance probably results from a decreased ability of the receptor to couple insulin binding to insulin action.

Adult↗
PubMed1981

Spontaneous tandem genetic duplications in Salmonella typhimurium arise by unequal recombination between rRNA (rrn) cistrons.

A method is described to detect and measure the frequency of spontaneous tandem genetic duplications located throughout the Salmonella genome. The method is based on the ability of duplication-containing strains to inherit two selectable alleles of a single gene during generalized transductional crosses. One allele of the gene carries an insertion of the translocatable tetracycline-resistance element Tn10; the other allele is a wild-type copy of that gene. Using this technique, we have measured the frequency of tandem duplications at 38 chromosomal sites and the amount of material included in 199 independent duplications. These results suggest that, in one region of the chromosome, tandem duplications are particularly frequent events. Such duplications have end points within rRNA (rrn) cistrons and probably arise by unequal cross-over between these dispersed repeated sequences. Spontaneously duplications of this type are harbored by as much as 3% of the bacterial population. Preliminary evidence suggests that such duplications may play a significant regulatory role under conditions of rapid growth. Our analysis has suggested the position on the genome of an additional rRNA cistron.

Base Sequence↗
PubMed1981

Direct demonstration of glycosylation of insulin receptor subunits by biosynthetic and external labeling: evidence for heterogeneity.

Insulin receptors of human lymphocytes (IM-9 line) were biosynthetically labeled with [3H]glucosamine, [3H]galactose, [3H]fucose, or [3H]mannose. After solubilization in Triton X-100, cell extracts were immunoprecipitated with serum from a patient containing autoantibodies to the insulin receptor. Na-DodSO4/polyacrylamide gel electrophoresis of the immunoprecipitates under reducing conditions showed the presence of major labeled subunits of apparent Mr 134,000 and 98,000 and a minor component of Mr 206,000. The ratio of activity in the 134,000 versus 98,000 Mr bands varied from 2:1 for mannose to 1.2:1 for galactose. In addition, the receptor subunits could be demonstrated when the cell surface of intact lymphocytes was labeled with NaB3H4 by using either the galactose oxidase (acts on nonreducing terminal galactose and N-acetylgalactosamine) technique or the periodate (oxidizes sialic acid) technique. With the periodate treatment, NaB3H4 labeled preferentially the Mr 98,000 band. With the galactose oxidase procedure, on the other hand, NaB3H4 labeled only the Mr 134,000 band; prior treatment with neuraminidase increased the labeling of this band and also revealed the Mr 98,000 subunit. These data demonstrate that the major subunits of the insulin receptor are complex glycoproteins that have differences in the nonreducing ends of the carbohydrate chains. In the Mr 134,000 subunit, there appear to be more exposed galactosyl or N-acetylgalactosaminyl (or both) residues, whereas the Mr 98,000 subunit appears to have a higher degree of sialylation. These labeling techniques provide new tools to examine the role of the carbohydrate moiety in insulin receptor function and turnover.

Cells, Cultured↗
PubMed1981

Insulin immunoreactive sites demonstrated in the Golgi apparatus of pancreatic B cells.

Insulin immunoreactive sites were localized in the Golgi apparatus of pancreatic B cells by light and electron microscopy. Identification of the Golgi apparatus by immunofluorescence required the prior degranulation of B cells with glibenclamide to reduce the insulin immunostaining due to secretory granules. In such cells, insulin immunofluorescence revealed brightly stained, crescent-shaped strands with form and location super-imposable on that of Golgi complexes seen in thin sections of the same cells. With the electron microscope, the insulin immunoreactive sites revealed by the protein A/gold technique were localized in the cisternae and vesicles of the Golgi apparatus of glibenclamide-treated and control B cells and over maturing and mature secretory granules. The quantitative evaluation of the intensity of the insulin immunoreactive sites in the Golgi apparatus revealed a density of sites 4 times more than cellular background values. The demonstration of insulin immunoreactivity in the Golgi apparatus provides direct evidence for the involvement of this compartment in the transport and maturation of proinsulin into insulin.

Animals↗
PubMed1981

Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue.

The recently developed low temperature embedding procedure with the resin Lowicryl K4M (Carlemalm E, Garavito M, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 656; Garavito M, Carlemalm E, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 658) was tested for its suitability for embedding of glutaraldehyde-fixed rat pancreatic tissue and for postembedding staining of thin sections with the protein A-gold (pAg) technique (Roth J, Bendayan M, Orci L: J Histochem Cytochem 26:1074, 1978) for amylase. Compared to conventional Epon embedding of glutaraldehyde fixed tissue, the low temperature embedding method with Lowicryl K4M resulted in a superior preservation of the general cellular fine structure, particularly in the Golgi apparatus. For low temperature embedded tissue, the quantitative evaluation of the immunocytochemical labeling for amylase showed a more specific staining of the rough endoplasmic reticulum, the Golgi apparatus, and the zymogen granules. This was due to a significant lowering of the background staining over all cellular organelles. The use of Lowicryl K4M at low temperature, due to the superior preservation, yields improved resolution and specificity in immunocytochemical postembedding staining.

Animals↗
PubMed1981

Increased 125I-insulin receptor binding to erythrocytes of hypoglycemic infants and children.

The measurement of 125I-insulin specific binding to erythrocytes obtained from seven infants and children with various hypoglycemic syndromes showed a significant increase in six patients with recurrent, documented, symptomatic hypoglycemia (percent specific 125I-insulin binding 9 to 14 versus 6.1 +/- 1.4% mean +/- 2 S.D. for 13 controls). The increase was due to an increased number of receptors sites per cell rather than to increased affinity for insulin. The patients included three children with nesidioblastosis, all after 90% pancreatectomy, two with leucine sensitivity, and two with glycogen storage disease type I. One of the patients with leucine sensitivity, who for 2 years before the study had no hypoglycemia, had normal insulin values (less than 10 microunits/ml). Thus, a symptomatic hypoglycemia correlated better with increased 125I-insulin binding than with plasma insulin values. Furthermore, Diazoxide therapy in two patients caused a mild but consistent decrease in the number of insulin receptor sites, and the institution of continuous nocturnal nasogastric feedings in a patient with glycogen storage disease type I was followed by amelioration of the hypoglycemia and a marked increase in 125I-insulin specific binding from 5.2 to 9.5%.

Adolescent↗
PubMed1981

Application of the protein A-gold technique for electron microscopic demonstration of polypeptide hormones.

The protein A-gold (pAg) technique enables the electron microscopic detection of antigenic material on thin sections of aldehyde-fixed and Epon-embedded tissue. Application of this technique to the endocrine pancreas and gastrointestinal mucosa using antisera against insulin, glucagon, somatostatin, pancreatic polypeptide, gastrin, and neurotensin allowed these polypeptides to be localized in the secretory granules of the corresponding specific cell types. The small size of marker gold particles permitting the precise identification of the labeled organelles coupled to the high specificity of the binding represent further evidence that the protein A-gold technique can be used as a general cytochemical reagent for the visualization of antigen-bound antibodies on thin sections.

Animals↗
PubMed1981

Plasma prolactin in acromegaly before and after treatment.

Plasma human prolactin (hPRL) was measured in 73 untreated acromegalic patients and was found to be elevated in 32% of the total population. Hyperprolactinemia was present in 40% of the females and in 27% of the male patients. In both groups, plasma hPRL correlated with plasma human growth hormone (hGH) levels with correlation coefficients of 0.38 (P less than 0.05) for females and 0.41 (P less than 0.005) for males. Forty-five patients were treated with conventional supervoltage pituitary irradiation and evaluated 2, 5, and 10 yr after treatment. The patients with hyperprolactinemia before irradiation showed a decrease in plasma hPRL at the most recent follow-up (mean +/- SEM, 125 +/- 34 vs. 67 +/- 15 ng/ml; P less than 0.01), although, in general, they did not achieve normal values. The patients who had normal plasma hPRL before irradiation (mean +/- SEM, 14 +/- 2 ng/ml) had increased levels after therapy (23 +/- ng/ml; P less than 0.005) but remained in the normal range during long term follow-up. In 10 patients followed for 1-10 yr without treatment, there was a tendency for plasma hPRL to rise progressively (mean increment, 122% above the initial value), with individual changes in hPRL strikingly parallel to the changes in plasma hGH. When serum hPRL was initially elevated, similar responses in both hormones were also seen in a small group of patients treated with surgical hypophysectomy. Galactorrhea was present in 5 of the 25 female patients; in 4 of the 5, plasma hPRL was within the normal range. Overall, these data suggest a closer relationship between hGH and hPRL in acromegaly than had been suspected, not only at the level of pituitary secretion but possibly also at the target cell.

Acromegaly↗
PubMed1981

Insulin in insects and annelids.

The fruitfly, Drosophila melanogaster, and the earthworm, Annelida oligocheta, were extracted with acid-ethanol by a classic method for recovering insulin from the pancreas. When each extract was filtered on a Sephadex G-50 column, a distinct peak of insulin immunoreactivity. The material in this peak had reactivity insulin (equivalent to 0.1 to 2 ng of insulin/g wet weight) was recovered in the region typical of insulin bioassay, measuring stimulation of glucose oxidation or lipogenesis by isolated rat adipocytes. The bioactivity was partially or largely neutralized by anti-insulin antibodies. In concordance with previous work showing the presence of material very similar to insulin in the blowfly and molluscs, we have confirmed the presence of insulin in insects and extended the observation to the earthworms. These findings suggest that insulin is more widespread in invertebrates than was previously thought. In a companion study (Proc. Natl. Acad. Sci. USA 77:6184-88, 1980), we have demonstrated material similar to insulin in unicellular organisms.

Adipose Tissue↗
PubMed1981

Insulin and insulin receptors in rodent brain.

While insulin effects on the central nervous system (CNS) mediated through hypoglycaemia are well known, direct insulin effects on the CNS remain controversial. Recently, we found insulin receptors in all areas of the rat brain, with highest concentrations in the olfactory bulb, cerebral cortex and hypothalamus; all areas involved in feeding. Insulin receptors in brain were, by multiple criteria, similar to insulin receptors on classical target tissues for insulin, such as liver and fat. Insulin itself has been identified in the rat brain at concentrations on average ten times higher than in plasma. Highest concentrations were found in the olfactory bulb and hypothalamus. Brain insulin was indistinguishable from purified insulin by its behaviour in the radioimmunoassay, radioreceptor assay, bioassay and gel chromatography. In two experimental models representing extremes of plasma insulin concentrations (obese hyperinsulinaemic mice and diabetic insulinopenic rats) there were no significant changes in the concentration of insulin receptors in brain while liver receptors were modified in the expected way. This may reflect the protective influence of the blood-brain barrier on some special quality of brain insulin receptors. Insulin concentrations in brain were also unchanged in both models, which is probably indicative of the local synthesis of insulin. The role of insulin in the CNS is unknown. Besides well known metabolic actions of insulin, new roles can be postulated such as neurotransmission, neuromodulation and paracrine signalling.

Animals↗
PubMed1981

[Dosimetry in case of whole-body irradiations with a linear accelerator (author's transl)].

The described whole-body irradiations are effected in order to prepare patients with acute myeloic leukemia for bone marrow transplantation. The patient is irradiated with equal doses from the right and the left side. So a dose of 10 Gy in the middle of the body is achieved which is determined individually by means of phantom measurements. In order to avoid the risk of an interstitial pneumonitis, the lung region is temporarily blinded in such a way that a dose of 8 Gy is obtained there in the middle of the body. During the irradiations, the accumulated dose is measured in different body regions with thermoluminescence dosimeters, a semi-conductor diode and, eventually, a ionization chamber. The scattering width is +/- 13%. We do our best to make the patient's position as comfortable as possible.

Bone Marrow Transplantation↗