Induction of autocrine epidermal growth factor receptor ligands in human keratinocytes by insulin/insulin-like growth factor-1

Autocrine activation of the epidermal growth factor (EGF) receptor on keratinocytes has been recognized as an important growth regulatory mechanism involved in epithelial homeostasis, and, possibly, hyperproliferative diseases. Insulin-like growth factor (IGF)-1 and insulin have been shown to be paracrine keratinocyte mitogens that bind to the type I IGF receptor which is expressed on actively proliferating keratinocytes in situ. In this report, we demonstrate that IGF-1/insulin induced production of keratinocyte-derived autocrine growth factors that bind to the EGF receptor. Increased steady-state mRNA levels for transforming growth factor alpha (TGF-α) and for amphiregulin (AR) were observed upon incubation of keratinocytes with mitogenic concentrations of IGF-1. IGF-1 also induced production and secretion of TGF-α and AR proteins as detected by immunoassays. An EGF receptor antagonistic monoclonal antibody abolished the mitogenic effect of IGF-1 on cultured keratinocytes. These results suggest that stimulation of keratinocyte growth by IGF-1 requires activation of an EGF receptor-mediated autocrine loop. © 1995 Wiley-Liss, Inc.     

Binding of an antagonistic monoclonal antibody to an intact and fragmented EGF-receptor polypeptide

A murine monoclonal antibody (No. 425) raised against human A431 carcinoma cells specifically immunoprecipitates the 170,000 molecular weight epidermal growth factor (EGF)-receptor from extracts of A431 cells as well as from extracts of human placenta and cultured fibroblasts, but does not recognize the murine receptor. Binding to the external domain of the human EGF-receptor was indicated by indirect immunofluorescent staining of fixed nonpermeable cells. The antibody binds to both glyco- and aglycoreceptor forms, indicating that the epitope is a part of the polypeptide chain. Binding of the antibody to the receptor is conformation dependent; i.e., denatured receptors lacking EGF-binding activity are not recognized by the antibody. The results of antibody binding studies indicate that the epitope is closely linked to the EGF binding active site, and is common to both high- and low-affinity EGF-receptors. Interaction of this epitope with the antibody inhibits EGF binding and bioactivity, and triggers receptor down-regulation, but does not generate EGFlike kinase-stimulatory or mitogenic responses either in vitro or in vivo. The antibody was tested for its ability to bind to domain-sized fragments of the 170-kDa EGF-receptor. It can recognize both the proteolytically generated 110-kDa EGF binding peptide, and a soluble 100-kDa EGF-receptor secreted by A431 cells. This indicates that the epitope recognized this antibody retains its conformation after proteolytic separation of the EGF binding domain from the rest of the receptor molecule.     

Enhancement of epidermal growth factor receptor expression on glioma cells by recombinant tumor necrosis factor α

Summary Recombinant tumor necrosis factor (rTNF; optimal dose 1000 U/ml) significantly increased the density of epidermal growth factor receptor (EGF-R) in three of four glioma cell lines in culture as determined by binding analysis of anti-EGF-R monoclonal antibody (mAb) 425. Since enhancement of EGF-R expression by rTNF- was inhibited when cells were treated with the protein synthesis inhibitor cycloheximide, the effects of rTNF may be protein-synthesis-dependent. The dose of rTNF that was optimal for up-regulation of EGF-R on glioma cells did not inhibit the growth of these cells.¹²⁵I-labeled mAb 425 lysed glioma cells in culture following its internalization into the cells. After glioma cells had been treated with rTNF, the growth-inhibitory effects of the mAb were significantly enhanced, probably a reflection of the increase in EGF-R density on the tumor cell surfaces. The rTNF effects were specific to the EGF-R and did not affect unrelated glioma-associated antigens. In our previous clinical trials,¹²⁵I-labeled mAb 425 showed immunotherapeutic effects in glioma patients. The present study provides the basis for considerations of combined immunotherapy of glioma patients with¹²⁵I-labeled mAb 425 and rTNF.     

Allocation of Photosynthate to Individual Tubers of Solanum tuberosum L.: I. RELATIONSHIP BETWEEN TUBER GROWTH RATE AND ENZYME ACTIVITIES OF THE STARCH METABOLISM

Potato plants (Solanum tuberosum L.) were grown in water culturein a controlled environment. Cooling (+8°C) of individualtubers decreased their growth rates and increased the growthrates of non-cooled tubers of the same plant. The carbohydrateconcentration in non-cooled and cooled tubers did not differsignificantly, but ¹⁴C-import from labelled photosynthate waslower in cooled than in non-cooled tubers. The markedly lowerconversion rate of ethanol-soluble ¹⁴C to starch in cooled,in comparison to non-cooled tubers, was not associated withsignificant differences in the in vitro activities of starchsynthase, ADPG-pyrophosphorylase and starch phosphorylase understandard assay conditions (+30°C). However, the Q₁₀-valuesof the enzymes differed in vitro in the temperature range between30°C and 8°C, leading to a marked decrease in the activityratio of ADPG-pyrophosphorylase/starch phosphorylase in cooledtubers. In tubers differing in growth rates without manipulation, 14d after tuber initiation significant positive correlations werefound between ¹⁴C-concentration of tuber tissue and the in vitroactivities of starch synthase and ADPG-pyrophosphorylase anda significant negative correlation between ¹⁴C-concentrationand starch phosphorylase. In contrast, in tubers which wereanalysed 5 d after initiation, there were only small differencesbetween tubers in growth rate, ¹⁴C import and the activity ratioADPG-pyrophosphorylase/starch phosphorylase. From various directand indirect evidence it is concluded that the growth rate ofindividual tubers, and thus the sink strength, is at least inpart controlled by the activity of starch synthesizing enzymes. Key words: Potato tuber, cooling, starch synthesizing enzymes     

First trimester prenatal diagnosis and detection of carriers of haemophilia A using the linked DNA probe DX13.

Although the use of a gene specific deoxyribonucleic acid (DNA) probe is the method of choice for detecting carriers of genes for rare genetic disorders, there will always be families in which such probes cannot be used because key subjects are not informative for restriction fragment length polymorphisms in or around the gene. In these cases closely linked DNA markers have to be used. An X chromosome specific DNA probe, DX13, which is closely linked to the haemophilia A locus on the X chromosome, was used for early prenatal diagnosis in two cases and to detect carriers in a series of nine possible heterozygote women. The first reported crossover between DX13 and the factor VIII:C locus was observed in this study. There are complexities inherent in using any linked DNA probe for assignment of genes, but such techniques are clinically important.     

Zur Frage der Wirkung von Morphiumsulfat auf das neurosekretorische System

Zusammenfassung Eine Belastung mit Morphium löst eine ADH- und Oxytocinmobilisierung aus. Durch Versuche an 38 mit Morphiumsulfatinjektionen behandelten Ratten wurde erwiesen, daß in gleicher Weise auch eine Neurosekretabnahme eintritt. Davon ist insbesondere die Neurohypophyse, das Abgabeorgan von Hypophysenhinterlappenhormonen und Neurosekret, betroffen. Bei Fortdauer der Stress-Situation, d. h. bei Fortsetzen der täglichen Morphiumsulfatinjektionen stellt sich eine Gewöhnung des neurosekretorischen Systems ein, die Neurosekreteinlagerung wird wieder auf den vorherigen Stand gebracht. Die Gewöhnung geht in der Regel so weit, daß vorbehandelte Tiere auch bei einer erheblichen Steigerung der Morphiumsulfatdosis keine Reaktion ihres neurosekretorischen Systems, d. h. keine Neurosekretabnahme, zeigen. Auch die fortgesetzte Belastung mit Morphiumsulfat führt demnach — wiedie Belastung mit Nikotinsäure — zur Ausbildung eines general adaptation syndrome im Sinne Selyes.

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Summary The administration of morphine to rats engenders a release of ADH and oxytocin. The injection of morphine sulfate to 38 rats shows that neurosecretory material depletion takes place in a similar manner, particularly in the posterior pituitary. After daily injections, the system adapts to the stress by redeposition of neurosecretory substances. The adaptation reaches the point where pretreated animals show no depletion after a much increased test dosis of morphine sulfate, similar to the prolonged stress with nicotinic acid. This is interpreted to represent adaptation in the sense used by Selye.

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Durchgeführt mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

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Die Vorversuche zu dieser Arbeit konnte der eine von uns (Rodeck) während seiner Gastassistentenzeit (1959) im Institut de Médecine et de Chirurgie expérimentales de l'Université de Montréal, Kanada (Direktor Prof. Dr. H. Selye) durchführen, wofür an dieser Stelle gedankt sei.     

Über die Alterung des neurosekretorischen Zwischenhirn-Systems in Zusammenhang mit der Regulation des Wasserhaushaltes

Zusammenfassung Der Alterungsprozeß der neurosekretorischen hypothalamo-neurohypophysären Regulationszentren des Wasserhaushaltes wurde an einem großen Kollektiv weißer Laboratoriumsratten verfolgt. Es konnte nachgewiesen werden, daß die Neurosekret- und damit die Hormonproduktion bis zum hohen Alter erhalten bleiben. Erst sehr alte Tiere zeigen eine mehr oder weniger deutliche Einschränkung der Neurosekretproduktion. Die Befunde werden mit den aus der Literatur bekannten Beobachtungen über die altersbedingten Veränderungen der Nieren in Zusammenhang gebracht. Wie während des Wachstums der Reifezustand der Niere auf allen Entwicklungsstufen der Reife der Hormonproduktionsstätten entspricht und beide wiederum mit den mit zunehmendem Alter sich weiter differenzierenden Erfordernissen des Allgemeinstoffwechsels koordiniert sind, so befinden sich auch im gesunden alternden Organismus neurosekretorisches Regulationszentrum, peripheres Erfolgsorgan (distaler Nierentubulus) und die mit dem Wasserhaushalt in Verbindung stehenden Stoffwechselprozesse dauernd im Zustand einer wohlausgewogenen Harmonie. Ein unkoordinierter Abbau eines Gliedes dieser Kette muß zwangsläufig zu Fehlregulation und Krankheit führen.Durchgeführt mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Audit of prenatal diagnosis for haemoglobin disorders in the United Kingdom: the first 20 years.

OBJECTIVES: To audit services for prenatal diagnosis for haemoglobin disorders in the United Kingdom. DESIGN: Comparison of the annual number of cases recorded in a United Kingdom register of prenatal diagnoses for haemoglobin disorders, with the annual number of pregnancies at risk of these disorders, by ethnic group and regional health authority. The number of pregnancies at risk was estimated using data on ethnic group from the 1991 census and data from the United Kingdom thalassaemia register, which records the number of babies born with thalassaemia. SETTING: The three national prenatal diagnosis centres for haemoglobin disorders. SUBJECTS: 2068 cases of prenatal diagnosis for haemoglobin disorders in the United Kingdom from 1974 to 1994. MAIN OUTCOME MEASURES: Utilisation of prenatal diagnosis by risk, ethnic group, and regional health authority. Proportion of referrals in the first trimester and before the birth of any affected child. RESULTS: National utilisation of prenatal diagnosis for haemoglobin disorders was around 20%. During the past 10 years it has remained steady at about 50% for thalassaemias and risen from 7% to 13% for sickle cell disorders. Utilisation for sickle cell disorders varies regionally from 2% to 20%. Utilisation for thalassaemias varies by ethnic group. It is almost 90% for Cypriots and ranges regionally for British Pakistanis from 0% to over 60%. About 60% of first prenatal diagnoses are done for couples without an affected child. Less than 50% of first referrals are in the first trimester. CONCLUSIONS: National utilisation of prenatal diagnosis for haemoglobin disorders is far lower than expected, and there are wide regional variations. A high proportion of referrals are still in the second trimester and after the birth of an affected child. The findings point to serious shortcomings in present antenatal screening practice and in local screening policies and to inadequate counselling resources, especially for British Pakistanis.