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J Roth

Publications and source records attributed to J Roth.

At least 559 records · Page 31Linked to original sources
PubMed1984

[Reproducibility of nonspecific bronchial reactivity].

The control of the non-specific bronchial reactivity (acetylcholine-test a 0.01% solution as ultrasonic aerosol) after a lapse of a year indicates that the findings of a bronchial hyperreactivity were reproducible in 32,6%, but the in the beginning observed inconspicuous reactivity in 92,9% of cases. In dependence upon a reproducible bronchial hyperreactivity manifest ventilation disturbances were significant more frequently ascertainable.

Acetylcholine↗
PubMed1983

The insulin-stimulated receptor kinase is a tyrosine-specific casein kinase.

Insulin stimulates a kinase that phosphorylates tyrosines in the insulin receptor; this kinase is tightly associated with the insulin receptor itself. We now show that the insulin-stimulated casein kinase, present in solubilized, lectin-purified receptor preparations from rat liver, is indistinguishable from the insulin receptor kinase. As with phosphorylation of the insulin receptor, insulin selectively enhanced by 2-3-fold the phosphorylation of tyrosines in casein. The insulin-stimulated activities of both kinases were inactivated at 37 degrees C with the same t0.5 of 5 min and were identically affected by alkylating agents. Both receptor and casein kinase activities were specifically coprecipitated by anti-receptor antibodies or by insulin and anti-insulin antibodies. When the latter type of immune complexes were incubated with an excess of insulin, both kinase activities were quantitatively recovered. We therefore conclude that insulin-stimulated receptor and casein phosphorylations are probably catalyzed by a single enzyme which is tightly associated with the receptor itself. Now, by replacing casein for receptor as substrate, it is possible to measure the enzymatic activity of this receptor-related kinase itself, i.e. independent of the receptor as substrate. Detection of this activity is improved in the presence of certain alkylating agents. Use of artificial substrates (in combination with alkylating agents) is particularly important to dissect the functional components of the receptor complex, to study mechanisms of enzyme regulation and especially in situations where the available receptor for study is limited, e.g. fresh or cultured cells from patients.

Amino Acids↗
PubMed1983

Insulin stimulates phosphorylation of serine residues in soluble insulin receptors.

Using lectin affinity-purified receptor preparations from human hepatoma cells, insulin (10(-7)M) specifically stimulated phosphorylation of the 95,000 dalton (beta) subunit of its own receptor. Phospho-amino acid analysis of the receptor subunit revealed that insulin increased at least 2.5-fold the content of phosphoserine and of phosphotyrosine. In intact cells, the major effect of insulin is to increase the phosphoserine content of its receptor. These findings are the first demonstration of an insulin-stimulated serine kinase in a cell-free system.

Carcinoma, Hepatocellular↗
PubMed1983

Insulin-stimulated phosphorylation of the insulin receptor precursor.

The alpha and beta subunits of the insulin receptor, Mr = 135K and 95K, appear to be synthesized via a single polypeptide precursor of Mr = 190K. We have investigated whether insulin stimulates the phosphorylation of this proreceptor, as is the case with mature receptor. Rat liver endoplasmic reticulum membranes were solubilized in Triton X-100 and chromatographed sequentially on wheat-germ agglutinin-agarose and lentil lectin-agarose columns. Phosphorylation of the lentil eluate with [gamma 32P]ATP revealed an insulin-stimulated phosphoprotein of Mr = 192K, which was recognized by antireceptor antibody, compatible with the receptor precursor. This suggests that further processing of the Mr = 190K insulin receptor precursor is not necessary for insulin binding, kinase activation, and receptor phosphorylation.

Animals↗
PubMed1983

Protein kinase activity of the insulin receptor in human circulating and cultured mononuclear cells.

In lectin-purified receptor preparations from human monocyte-like cell (U-937), insulin (10(-7)M) stimulated phosphorylation of the 95,000 dalton subunit of its own receptor. In addition, insulin stimulated phosphorylation of exogenously added substrates like casein, (T,G)-A--L, and histones. Phosphorylation of the synthetic peptide (T,G)-A--L indicates the presence of at least one insulin-dependent tyrosine kinase in these cell extracts. Insulin receptor preparations from freshly isolated human mononuclear blood cells were also shown to possess insulin-dependent casein and (T,G)-A--L kinase activity. Phosphorylations in these systems are specific for insulin and dependent on insulin concentration. A simple and rapid method is described that is relevant for clinical investigations of early postbinding events.

Cells, Cultured↗
PubMed1983

Insulin stimulated phosphorylation of its own receptor. Activation of a tyrosine-specific protein kinase that is tightly associated with the receptor.

In solubilized, (wheat germ) lectin-purified preparations of rat liver membranes, insulin stimulated the incorporation of 32P from [gamma-32P]ATP into tyrosine residues of insulin receptor, casein, and histones. Despite the presence of both protein kinase and phosphatase activities in these preparations, no decrease in the 32P content of receptors (preincubated with or without insulin (0.5-100 nM)) was detected whether 32P incorporation was terminated by excess ATP, ATP + Mn2+, EDTA, or phosphatase inhibitors. Similarly, there was no decrease in the 32P content of phosphoreceptors incubated for up to 60 min with fresh receptor preparations in the presence or absence of insulin. Dephosphorylation of the insulin receptor to 20% of original 32P content only occurred when alkaline phosphatase was added to the preparations. It is concluded that endogenous receptor phosphatase(s) are either missing or inactive in these preparations, and consequently, insulin stimulates phosphorylation of its own receptor by activating a protein kinase. The kinase activity is tightly associated with the receptor itself; insulin also stimulated the phosphorylation of both receptor subunits in purified insulin-receptor complexes that had been immunoprecipitated by anti-insulin antibodies. However, the phosphorylating machinery is much more sensitive to heat inactivation than the binding function (90% less 32P incorporation versus 15% less binding during 60-min incubation at 37 degrees C), suggesting that the kinase is not associated exclusively with the insulin-binding domain.

Animals↗
PubMed1983

Characterization of insulin-mediated phosphorylation of the insulin receptor in a cell-free system.

Insulin stimulates phosphorylation of both alpha- and beta- subunits of its own receptor in a cell-free system. A solubilized lectin-purified preparation of insulin receptors from rat liver membranes was preincubated with or without insulin at 4 degrees C and labeled for 10 min with Mn[gamma- 32P]ATP; the receptor subunits were isolated by specific immunoprecipitation with anti-receptor antibodies, followed by gel electrophoresis in sodium dodecyl sulfate. In gels run under reduced conditions, two bands (Mr = 135,000 and 95,000) were selectively labeled. These correspond exactly to the position of the alpha- and beta-subunits of the insulin receptor. Labeling of the Mr = 95,000 band was approximately 5-fold that of the Mr = 135,000 band. No labeled bands were detected when identical samples were immunoprecipitated in control serum. Phosphorylation of the receptor subunits required the presence of the divalent cation Mn2+ or Co2+; other cations such as Mg2+, Cr3+, Ca2+, and Zn2+ were ineffective. [gamma- 32P]ATP served as the 32P donor, whereas [gamma- 32P]GTP was ineffective. Phosphorylation of both subunits was stimulated 4-6-fold after a 60-min exposure to 10(-7) M pork insulin. Insulin-stimulated phosphorylation was half-maximal after 5 min of incubation with 10(-7) M insulin or after 18 h with 3 X 10(-10) M hormone. The enhanced phosphorylation was specific for insulin and its analogs; guinea pig insulin was about 2% as potent as pork insulin, whereas epidermal growth factor, adrenocorticotropic hormone, and glucagon, as well as cAMP, were ineffective. The rapidity and specificity of this reaction, as well as the presence of all necessary components in the plasma membrane, suggest that insulin-mediated receptor phosphorylation is one of the earliest biochemical steps following insulin binding.

Adenosine Triphosphate↗
PubMed1983

Size and erosive features of the sella turcica in acromegaly as predictors of therapeutic response to supervoltage irradiation.

Basal growth hormone levels and the sella turcica of patients with acromegaly were evaluated. Fifty of these patients were followed up, with fasting growth hormone levels determined at several intervals within 10 or more years after supervoltage pituitary irradiation. Prior to therapy, basal growth hormone levels were positively correlated with an estimate of tumor size, as reflected by sella abnormalities. Sella abnormality criteria, developed by Hardy et al., were used as the correlating factor. The percentage of fall in growth hormone levels after radiotherapy was indistinguishable in these patients, regardless of sella grade. However, since the larger, more erosive, tumors were associated with higher pre-therapy plasma growth hormone levels, the median growth hormone levels were higher at various intervals after treatment of this group. We suggest that the size and erosive features of the bony sella offer a crude, but possibly useful, predictor of response to supervoltage irradiation in acromegaly.

Acromegaly↗
PubMed1983

Quality control of radiotherapy in acute lymphocytic leukemia protocol treatment: experience with 610 cases.

Quality assurance programs are necessary in multi-institutional cooperative group clinical trials to ensure that possible inter-institutional differences in selection, treatment and evaluation of patients will not erode the statistical assessment of these clinical trials. The Radiotherapy Committee of the Cancer and Leukemia Group B examined the evaluability and appropriateness of treatment of patients entered into two protocols for childhood acute lymphocytic leukemia, 7411 prior to and 7611 after the development of a quality assurance review program. Of the 348 patients entered into 7411, 37% were evaluable and 26% were appropriately treated in 1974 when the protocol opened. This rose to 53 and 35% in the last year of the study. On the other hand, in 7611 with an ongoing quality assurance program, the evaluability rate initially was 63% and rose to 73% and the appropriateness rate rose from 37 to 61%. This change in performance which was statistically significant at the P = 0.001 level is attributed to the impact of the Quality Assurance Review Center correspondence. Improvement in performance occurred almost entirely in the principal centers and not in satellite institutions. This difference in performance was statistically significant at the P = 0.05 level, indicating that adherence to protocol requirements increases with increased participation in studies.

Child↗
PubMed1983

Regional distribution of N-acetyl-D-galactosamine residues in the glycocalyx of glomerular podocytes.

Helix pomatia lectin (HPL) bound to colloidal gold was used as a specific cytochemical probe for the localization of terminal nonreducing N-acetyl-D-galactosamine residues in thin sections of rat kidney. In the glomerulus, lectin-binding sites were associated only with the podocyte foot process bases and were not found on the free cell surface of podocytes or on any other glomerular components. Gold-particle label was often arranged in the form of clusters which extended from the foot process base to the lamina rare externa and lamina densa of the basement membrane. In contrast, wheat germ lectin (WGL)-binding sites (beta-[1 leads to 4] linked N-acetyl-D-glucosamine residues and N-acetylneuraminic acid residues) were found in all regions of the podocyte plasma membrane and on the cell surface of all other glomerular cell types. In addition, WGL-binding sites were present in all three layers of the glomerular basement membrane (GBM) as well as in the mesangial matrix. A quantitative evaluation of the pattern of labeling for HPL-binding sites together with the sugar specificity of this lectin suggest that a component of the glycocalyx is being detected rather than a basement membrane component. This was confirmed by the absence of H. pomatia lectin-binding sites in preparations of isolated GBM which retained, however, wheat germ lectin-binding sites. These data show that the glycocalyx of the foot process base is a highly specialized cell surface domain with respect to its carbohydrate composition.

Acetylgalactosamine↗
PubMed1983

Water removal and solute additions determining increases in renal medullary osmolality.

Osmolality and solute concentrations of the mammalian renal medulla increase and decrease with changing urine osmolality. These changes are brought about by addition or removal of solute or water to or from the renal medullary tissue. In Munich-Wistar rats and Syrian hamsters, males and females, actual amounts of and the various solutes involved in these changes were determined. Kidneys were removed from animals killed in different stages of water diuresis and antidiuresis. The renal medulla was analyzed by a new method that permits determination of water and solutes on the same piece of tissue. Removal of water and addition of urea were the two most important factors in raising inner medullary osmolality. Papillary water content was inversely related to the papillary osmolality and was 50% lower in extreme antidiuresis compared with water diuresis. Rats had higher papillary water content than hamsters. In the outer medulla, water removal was significant in the hamsters but not in the rats. Addition of urea to the papillary tissue exceeded the osmotic equivalent of NaCl by a factor of 2.8 in both rats and hamsters. Females of both species showed greater changes than males in amounts of urea in the inner medulla.

Animals↗
PubMed1983

Immunohistochemical localization of carbonic anhydrase in postnatal and adult rat kidney.

With use of specific antibodies against human and rat erythrocyte carbonic anhydrase C and human carbonic anhydrase B, only the isozyme C could be detected by immunofluorescence in rat kidney epithelial cells. In the postnatal kidney a few cells were positive after 2 days, but the number of fluorescent cells increased during the first few weeks of life to reach the final adult levels after 3 wk in the cortex and 5 wk in the medulla. In the postnatal and adult kidney a characteristic mosaic pattern of fluorescence was seen in the late distal tubule, the connecting segment, and the collecting tubule, where the mitochondria-rich dark cells were brightly fluorescent. In addition, in later postnatal stages and in the adult, the entire epithelium of the initial portion of descending thin limbs of Henle (long loops) was labeled. Some kidney regions that had previously been shown to contain carbonic anhydrase activity by biochemical and histochemical techniques stained only weakly with the immunocytochemical method. This suggests either that the enzyme in these regions does not cross-react strongly with our antibodies or that these regions contain only low amounts of carbonic anhydrase, at the limit of the detection threshold of our techniques.

Aging↗
PubMed1983

Applications of immunocolloids in light microscopy. II. Demonstration of lectin-binding sites in paraffin sections by the use of lectin-gold or glycoprotein-gold complexes.

A new procedure is presented for the light microscopic demonstration of specific sugar sequences of oligosaccharides in glycoconjugates by lectins combined with the colloidal gold marker system. Tissue sections from aldehyde-fixed and paraffin embedded rat kidney were stained either in a one-step method with lectin directly bound to particles of colloidal gold or in a two-step method using non-labeled lectin and glycoprotein labeled with colloidal gold. In both methods the presence of lectin-binding sites in the tissue sections is revealed by the appearance of a red coloration that is due to the accumulation of gold particles. The high specificity of the technique is combined with a good sensitivity and resolution as demonstrated by a differential plasma membrane staining in renal epithelial cells. The lectin-gold or glycoprotein-gold complexes remain stable for months and produce a permanent nonbleaching staining.

Animals↗
PubMed1983

Application of lectin--gold complexes for electron microscopic localization of glycoconjugates on thin sections.

A method is described for the electron microscopic detection of lectin-binding sites in different cellular compartments and extracellular structures that uses thin sections from resin-embedded tissues. Various lectins (Ricinus communis lectin I and II, peanut lectin, Lotus tetragonolobus lectin, Ulex europeus lectin I, Lens culinaris lectin, Helix pomatia lectin, and soybean lectin) were bound to particles of colloidal gold and used for direct staining of thin sections or glycoprotein--gold complexes were prepared and applied in an indirect technique (concanavalin A and horseradish peroxidase--gold complex; wheat germ lectin and ovomucoid--gold complex). The details for preparation of such complexes from 14 nm gold particles are reported. The conditions of tissue processing that gave satisfactory staining results and good fine structure preservation were mild aldehyde fixation without osmification and low temperature embedding with the hydrophilic resin Lowicryl K4M. None of the so-called etching procedures was necessary prior to labeling of Lowicryl K4M thin sections. Examples of the use of this approach for detection of glycoconjugates in the rough endoplasmic reticulum, Golgi apparatus, and mucin of intestinal goblet cells as well as plasma membrane and various intracellular structures of absorptive intestinal and renal tubular cells are shown. A comparison is made with preembedding staining results on Concanavalin A-binding site localization in rat liver which shows that problems of penetration common in such a technique are circumvented by the postembedding approach described here. Concanavalin A-binding sites were not only consistently found in nuclear envelope, rough and smooth endoplasmic reticulum, plasma membranes, and collagen fibers, but also in mitochondria, glycogen, ribosomes, and nucleus. These data and those of a previous investigation (Roth J, Cytochem 31:547, 1983) prove the applicability of this cytochemical technique for postembedding localization of glycoconjugates by light and electron microscopy.

Animals↗
PubMed1983

Decreased insulin binding to cultured cells from a patient with the Rabson-Mendenhall syndrome: dichotomy between studies with cultured lymphocytes and cultured fibroblasts.

[125I]Insulin binding has been studied with cultured cells from an insulin-resistant patient with the Rabson-Mendenhall syndrome. Previously, this patient has been demonstrated to have decreased insulin binding to circulating blood cells. Similarly, [125I]insulin binding to cultured lymphocytes and cultured fibroblasts was markedly decreased. Whereas the decrease in [125I]insulin binding to cultured lymphocytes appeared to result from a decrease in receptor number in cultured lymphocytes, there appeared to be a reduction in the affinity of [125I]insulin binding to cultured fibroblasts. This discrepancy between the observations with the two cell types is a general phenomenon which has been described in patients with a variety of syndromes of extreme insulin resistance. It is possible that the interpretation of the studies with the fibroblasts is complicated by the confounding influence of receptors for insulin-like growth factors on the surface of those cells. If the number of insulin receptors is sufficiently reduced on fibroblasts from the patient with extreme insulin resistance, then the low level of residual [125I]insulin binding may involve primarily receptors for insulin-like growth factors, which bind insulin with relatively low affinity.

Adolescent↗