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Biomedical subjects

S Jacobs

Publications and source records attributed to S Jacobs.

At least 163 records · Page 9Linked to original sources
PubMed1986

Purification of insulin-like growth factor I receptor from human placental membranes.

Insulin-like growth factor (IGF) I receptor was purified from Triton X-100-solubilized human placental membranes by wheat germ agglutinin-Sepharose chromatography followed by immunoaffinity chromatography using alpha IR-3, a monoclonal antibody directed against the IGF-I receptor. Purification of 3200-fold and 2800-fold was achieved from wheat germ agglutinin-Sepharose eluates with regard to IGF-I binding and kinase activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed two major protein bands corresponding to the alpha and beta subunits of the receptor, which accounted for at least 90% of the protein content. The purified receptor bound 10-20 micrograms of IGF-I/mg of protein and was more than 95% free of contamination by insulin receptor. It sedimented in glycerol gradients as a single species with a sedimentation coefficient of 13.7 S and gave three protein bands with Mr = approximately 300,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, indicating that alpha 2 beta 2 is an intact form of the IGF-I receptor. The purified receptor, when incubated with [gamma-32P] ATP, became phosphorylated at tyrosine residues of its beta subunit. This was stimulated 3-fold by IGF-I. It also had IGF-I-stimulated tyrosine kinase activity (5264 pmol of 32P incorporated/min/mg of protein) toward a synthetic peptide corresponding to the autophosphorylation site of pp60src. These data strongly suggest that it is a tyrosine-specific protein kinase.

Antibodies, Monoclonal↗
PubMed1986

Phosphorylation of receptors for insulin and insulin-like growth factor I. Effects of hormones and phorbol esters.

The phosphorylation of receptors for insulin and insulin-like growth factor I was studied by phosphoamino acid analysis and tryptic phosphopeptide maps in an attempt to determine if protein kinase C is involved in their phosphorylation in response to insulin and insulin-like growth factor I, respectively. Two cell lines were utilized, Hep G2 and IM-9 cells. sn-1,2-Dioctanoylglycerol and 12-O-tetradecanoylphorbol 13-acetate (TPA), agents known to activate protein kinase C, stimulated the phosphorylation of the beta subunits of both receptors, as did their hormones. In unstimulated cells, phosphorylation of the insulin receptor occurred on seryl and to a lesser extent on threonyl residues. TPA stimulated seryl and threonyl phosphorylation that resulted in the appearance of four major phosphoserine-containing phosphopeptides which were not detected in the basal state and an increase in phosphorylation of a phosphothreonine-containing peptide which was present in the basal state. Insulin treatment resulted in the appearance of three major phosphotyrosine-containing tryptic peptides. In IM-9 cells, insulin also increased the phosphoserine and possibly the phosphothreonine content of the beta subunit. In both cells, the major phosphoserine-containing peptides that were stimulated by TPA were not detected following treatment with insulin. Very similar results, including similar peptide maps, were obtained for the insulin-like growth factor I receptor from cells treated with TPA and insulin-like growth factor I. Although not entirely conclusive, these results suggest that the insulin- and insulin-like growth factor I-stimulated phosphorylation of their receptors does not result from activation of protein kinase C.

Carcinoma, Hepatocellular↗
PubMed1986

Complete glycosylation of the insulin and insulin-like growth factor I receptors is not necessary for their biosynthesis and function. Use of swainsonine as an inhibitor in IM-9 cells.

Swainsonine, an indolizidine alkaloid which is a potent inhibitor of the Golgi enzyme, mannosidase II, leads to the production of incompletely processed glycoproteins lacking complex type oligosaccharides. This inhibitor has been used to examine the importance of terminal sugar groups in the biosynthesis and function of both the insulin receptor and the insulin-like growth factor I receptor. IM-9 cells were metabolically labeled using [35S]methionine and the two receptors were independently immunoprecipitated using specific monoclonal antibodies. The incompletely processed receptors have slightly lower molecular weights and contain hybrid rather than complex type oligosaccharides as indicated by their sensitivity to endoglycosidase H and neuraminidase. Both receptors made in the presence of swainsonine are still autophosphorylated in the presence of the respective hormone. The insulin receptor made in the presence of the inhibitor can be affinity labeled at the cell surface using 125I-insulin and disuccinimidyl suberate cross-linking; there is also no significant difference in its affinity for insulin. These results suggest that for the insulin and insulin-like growth factor I receptors to be synthesized, processed, and function normally, they do not require all of the sugars which are normally added in the terminal stages of glycosylation.

Affinity Labels↗
PubMed1986

Psychological distress, depression and prolactin response in stressed persons.

We examined the relationship of psychological distress to serum prolactin response in 54 persons who had lost a spouse or were threatened with a loss. We found that our two measures of psychological distress, both separation anxiety and depression, were directly correlated with prolactin response during a stressful interview (p less than .05). When we stratified the sample first by depression score and then by separation anxiety, we found a positive correlation between separation anxiety and prolactin response only in the highly depressed half of the sample (r = .32) and a positive correlation between depression and prolactin response only in the highest quartile of intensity for separation anxiety (r = .49, p less than .05). This suggested that both depression and separation anxiety, each in conjunction with high levels of the other but not independently, rendered the individual under stress more physiologically sensitive to distressing challenges such as a stressful interview. Alternatively, it was global distress above a certain threshold that was associated with degree of physiological response.

Anxiety, Separation↗
PubMed1986

Newborn screening for hemoglobin abnormalities. A comparison of methods.

Peripheral blood from 1,000 newborn infants of black (641) and Southeast Asian (359) ancestry were screened for hemoglobin variants. Results obtained from the combination of cellulose acetate (CAC) and citrate agar (CAG) electrophoresis were compared with isoelectric focusing (IEF) electrophoresis. There was complete agreement between the two methods on assignment of Hb S trait, Hb C trait, Hb E trait, and homozygous Hb E. IEF identified small amounts of Hb A in two newborn infants with Hb S-beta + thalassemia; the CAC and CAG electrophoretic patterns were indistinguishable from sickle cell anemia. One hundred twenty newborn infants with Hb Bart's were detected by IEF; 51 of these were found on CAC. Although IEF was more sensitive in detecting small amounts of hemoglobin, it is not clear if the improvement in detection warrants adopting this form of electrophoresis for routine screening of newborn infants.

Electrophoresis↗
PubMed1986

The measurement of grief: age and sex variation.

Age and sex variation in the expression of grief were examined using a structured assessment to test clinically based impressions that widowers by comparison with widows and older bereaved persons by comparison with middle-aged counterparts would report distinctive aspects of grief and less intense distress. One hundred and fourteen acutely bereaved spouses were interviewed one month after a loss. The psychological assessment used was designed to measure several dimensions of grief including separation anxiety, numbness-disbelief and depression. Widows reported significantly more numbness-disbelief and depression than widowers but no difference in the intensity of separation anxiety. Contrary to our hypotheses, no attenuation in the intensity of grief was observed among older bereaved spouses, though they reacted to the loss with less emotional numbness and disbelief of the reality than the middle-aged comparison group. Furthermore, they reported no more neurovegetative or illusory symptoms than middle-aged, bereaved spouses. Older widowers experienced more separation anxiety than younger widowers. It seems possible that these differences account, in part, for age and sex variation in the morbidity and mortality of bereavement.

Age Factors↗
PubMed1986

Interaction of the monoclonal antibodies alpha IR-1 and alpha IR-3 with insulin and somatomedin-C receptors.

alpha IR-3, a monoclonal antibody that interacts with the somatomedin-C receptor, inhibited the binding of somatomedin-C, but not of insulin, to human placental membranes and intact IM-9 cells. alpha IR-1, a monoclonal antibody that interacts with the insulin receptor, did not inhibit the binding of either hormone. Inhibition of somatomedin-C binding by alpha IR-3 was mainly due to a decrease in its affinity. 125I-Labeled alpha IR-3 bound specifically to placental membranes and intact IM-9 cells and was inhibited by concentrations of unlabeled alpha IR-3 that were lower than those required to inhibit somatomedin-C binding. [125I]alpha IR-3 binding was also inhibited by somatomedin-C and insulin, but only at very high concentrations. A410, a rabbit antiserum that reacts with both receptors for insulin and somatomedin-C, also inhibited labeled alpha IR-3 binding. alpha IR-I did not. These results help to define the epitopes with which these antibodies interact.

Animals↗
PubMed1985

Mechanism of phorbol diester-induced regulation of surface transferrin receptor involves the action of activated protein kinase C and an intact cytoskeleton.

Phorbol diesters are tumor-promoting agents that cause differentiation of HL60 human leukemic cells and concomitantly regulate surface transferrin receptors. Regulation of transferrin receptors by phorbol diesters involves receptor internalization in association with increased receptor phosphorylation (hyperphosphorylation). The intracellular mechanism of action of phorbol diester involves binding to and activation of the Ca2+-phospholipid-dependent protein kinase (protein kinase C). Present studies comparing results obtained with whole cells and those from a cell-free system reconstituted from purified protein kinase C and transferrin receptor components have revealed that the transferrin receptor is phosphorylated by protein kinase C activated by phorbol esters. Following tryptic digestion and two-dimensional separation of phosphopeptides of phosphorylated transferrin receptors, two major and several minor phosphoserine-containing fragments are resolved. These fragments are identical whether transferrin receptor is phosphorylated in whole cells incubated with phorbol diesters or following phosphorylation of affinity immobilized transferrin receptor in the in vitro reconstitution system. Phosphoamino acid analysis of these fragments indicates that serine is the only amino acid phosphorylated in whole cells or in the cell-free system. In addition, colchicine is shown to inhibit in a dose-dependent manner phorbol diester-induced internalization but not hyperphosphorylation of the surface transferrin receptor in whole cells. This inhibition is specific for colchicine since inactive beta- and gamma-Lumicolchicine have no such effect, while taxol reverses the inhibition. These results indicate that the phorbol diester-mediated process of down-regulation of the surface transferrin receptor is associated with phosphorylation of the receptor by activated protein kinase C and requires an intact cytoskeleton to affect receptor internalization.

Cell Line↗
PubMed1985

Changes in the respiratory gas tensions of a pneumonectomy space and their application to the diagnosis of bronchopleural fistula.

The oxygen (pO2) and carbon dioxide (pCO2) gas tensions of a pneumonectomy space were studied in an animal model. The 2 gases were measured in a standard blood gas analyser. The space gas pO2 and pCO2 equilibrated to a steady state within 48 hours of thoracotomy (pO2 mean 53.9 +/- 9.3 SD torr; pCO2 mean 44.8 +/- 9.9 SD torr). In the presence of a bronchopleural fistula (BPF), the pO2 rose significantly (mean pO2 118.4 +/- 13.2 SD torr; p less than 0.001). The space pCO2 usually fell, but the response was inconsistent (mean pCO2 33.6 +/- 16.4 SD; p = NS). A further rise in space pO2 could sometimes be induced by allowing the animals to breathe 100% oxygen by face mask. Data from 4 clinical cases of BPF support the concept that measurement f the space gas pO2 could be used as a bedside test for BPF.

Animals↗
PubMed1985

Cellular receptor for 125I-labeled tumor necrosis factor: specific binding, affinity labeling, and relationship to sensitivity.

Tumor necrosis factor (TNF) is a proteinaceous toxin shed by stimulated myeloid cells. Murine TNF was radioiodinated to a specific activity of 1 mCi/nmol (1 Ci = 37 GBq) of monomer. 125I-labeled TNF (125I-TNF) retained complete cytotoxic activity and it was immunochemically identical to the native toxin in a quantitative immunoprecipitation assay. It could be shown by competition binding that 125I-TNF bound to intact L929 cells with a specificity equal to that of native toxin. The conditions of time, temperature, and concentration involved in equilibrium specific binding to intact cells were studied in detail. When binding was carried out at 4 degrees C for 18 hr, four cell lines sensitive to 125I-TNF cytotoxicity demonstrated high-affinity binding. The binding reached half-maximal level at 3 pM and saturated at 30 pM. These concentrations approximated those required for cell death. Scatchard analysis gave approximately 1000 sites per cell. J774.1 cells, the source of the toxin, demonstrated similar binding but were not sensitive to 125I-TNF cytotoxicity. Other sensitive cell lines and freshly extracted tumor cells showed specific binding at 3 pM. Normal lymphoid organ cell suspensions and two human tumorigenic cell lines were not sensitive and failed to demonstrate specific binding. 125I-TNF, covalently cross-linked to its receptor on sensitive L-M cells with disuccinimidyl suberate, was isolated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography. Two specific bands were identified. The most prominent band had a mobility corresponding to a molecular mass of 95 kDa and the second band had a molecular mass of 75 kDa. The presence of the binding site appears to be necessary but not sufficient to explain the sensitivity of cells to the cytotoxic action of TNF.

Affinity Labels↗
PubMed1985

Acute bereavement, threatened loss, ego defenses and adrenocortical function.

In this study of 43 acutely bereaved widows and widowers and a comparison group of 24 nonbereaved married persons who experienced critical illness in a spouse, we found no evidence of higher levels of affective arousal, higher levels of adrenocortical activity, less effective ego defenses, or higher levels of defensive work among bereaved persons by comparison with nonbereaved persons. Furthermore, we failed to observe expected relationships between ego defensive function and adrenocortical activity among either bereaved or nonbereaved persons. These observations are consistent with one previous study of bereaved parents.

Arousal↗
PubMed1985

Evidence from monoclonal antibody studies that insulin stimulates deoxyribonucleic acid synthesis through the type I somatomedin receptor.

Somatomedin-C/insulin-like growth factor-I (Sm-C/IGF-I) and insulin stimulate DNA synthesis and cell replication in cultured human fibroblasts. It has been postulated that the growth-promoting actions of both peptides are mediated through the type I Sm-C/IGF-I receptor. This study tests this hypothesis using two recently developed monoclonal antibodies. The antibody designated sm 1.2 is directed to Sm-C, whereas the antibody designated alpha IR-3 is directed against the type I receptor for Sm-C/IGF-I. Radiolabeled monoclonal antibody alpha IR-3 was bound to human foreskin fibroblasts in a reversible time-dependent fashion, with 90% of the specific binding complete after 6 h of incubation at 15 C. Binding of [125I]alpha IR-3 was completely inhibited by excess unlabeled antibody, but not by 50 nM Sm-C or 1000 nM insulin. Specific binding of [125I]Sm-C fell to 27% of the control value in the presence of 50 nM alpha IR-3, and this concentration of antibody significantly reduced the mitogenic response to both Sm-C and insulin. Antibody sm 1.2 blocked the mitogenic response to exogenous Sm-C, but did not block the response to insulin; indeed, in some experiments, sm 1.2 enhanced the response to insulin. We postulate that this enhancement is the result of neutralizing endogenously produced Sm-like substances. This study provides further evidence that the growth-promoting effects of insulin in this cell type are the result of interaction with the Sm-C/IGF-I receptor.

Antibodies, Monoclonal↗
PubMed1985

Human insulin receptor gene. Data supporting assignment to chromosome 19.

Somatic cell hybrid clones constructed by crossing human skin fibroblasts with mouse L cells have been examined for expression of human insulin receptors, using a monoclonal antibody directed against the human insulin receptor. Data obtained in this study support the assignment of the human gene for the insulin receptor to chromosome 19.

Animals↗
PubMed1984

Analysis of the insulin receptor by anti-receptor antibodies and flow cytometry.

We characterized insulin receptors on a human lymphoblastoid cell line (IM-9) and studied their regulation using anti-receptor antibodies and fluorescence flow cytometry. The fluorescence intensity distribution of insulin receptors on cells was determined by incubating the cells with one of three different anti-receptor antisera (human serum B-9 containing polyclonal autoantibodies, serum from a rabbit with polyclonal antibodies, and a monoclonal antibody to the receptor produced in mouse hybridomas), followed by incubation with an appropriate fluorescein isothiocyanate-labeled second antibody and analysis on an Epics-V flow cytometer. All three anti-receptor antibodies specifically labeled the insulin receptors. The monoclonal antibody showed the highest level of labeling. Treatment of cells with proteolytic enzymes, such as trypsin or chymotrypsin, produced a dose-dependent loss of 125I-labeled insulin (125I-insulin) binding but a relatively small decrease in the binding of anti-receptor antibodies, suggesting that most antibody binding occurred in domains other than the insulin binding site. Treatment with glycosidic enzymes, such as neuraminidase and beta-galactosidase did not affect the binding of 125I-insulin, and fluorescence was actually enhanced by about 20% in the beta-galactosidase-treated cells. Exposure of IM-9 cells to insulin resulted in a reduction in the number of insulin receptors. Analysis of the down-regulated cells by immunofluorescence revealed a complete correlation between the percent binding of 125I-insulin and percent peak fluorescence. In all cases, receptors were lost proportionally from all cells, yielding a single symmetrical peak by fluorescence analysis. Exposure of IM-9 cells to anti-receptor antibodies at 37 degrees C for 16 hr also produced a down-regulation in the number of insulin receptors. Incubation with human antiserum B-9 caused a 95% loss of both 125I-insulin binding and peak fluorescence, while the monoclonal antibody resulted in a 50% loss of receptors. Incubation of cells with anti-receptor antibodies for 2 hr at 4 degrees C did not produce any receptor loss; however, the human anti-receptor antisera B-2 and B-9 inhibited the binding of the monoclonal anti-receptor antibody by about 50%, suggesting that these antisera contained autoantibodies directed at the monoclonal antibody binding site. These data indicate that insulin receptors can be regulated by both insulin and anti-receptor antibody and demonstrate the utility of immunofluorescence and flow cytometry as a tool for the study of the insulin receptor.

Antibodies, Monoclonal↗
PubMed1984

Association of phorbol ester-induced hyperphosphorylation and reversible regulation of transferrin membrane receptors in HL60 cells.

Phorbol diesters are tumor-promoting agents that cause differentiation of HL60 human leukemic cells and concomitantly alter surface transferrin-receptor expression [Rovera, G., Ferreo, D., Pagliardi, G. L., Vartikar, J., Pessano, S., Bottero, L., Abraham, S. & Lebman, D. (1982) Ann. N.Y. Acad. Sci. 397, 211-220]. Transferrin-receptor regulation is shown here to result from a rapid and reversible internalization process that is temporally associated with reversible increased phosphorylation (hyperphosphorylation) of the transferrin receptor. Such a reversible mechanism involving regulation of these surface proteins could result in the rapid generation of an early signal for HL60 cellular differentiation.

Carcinogens↗