Immunohistochemical localisation and developmental aspects of epidermal growth factor in the rat

The tissue localisation and time of first appearance of Epidermal Growth Factor (EGF) in the developing rat were investigated by means of immunohistochemistry, radioimmunoassay and radioreceptor assay. In this study we were able to show, that EGF appears prenatally in the lung and the kidney from gestational day 19. Postnatally EGF was found also in the gastrointestinal tract, first in Brunner's glands of the duodenum (at birth), next in the Paneth cells (day 7), and finally in the submandibular glands (day 25). The immunohistochemical and radioreceptor results are consistent, whereas the radioimmunoassay detects EGF later and in smaller quantities, than does the radioreceptor assay. These differences will be discussed.     

Detection of endogenous epidermal growth factor-like activity in the developing chick embryo

Epidermal growth factor-like activity has been detected by radioreceptor assay and radioimmunoassay in the developing chick embryo. Very little activity could be detected prior to Day 8 of embryonic life (hatching is at Day 21). A peak of EGF activity was detectable by both assays over Days 10 to 12. The EGF activity then fell to virtually undetectable levels during Days 14 to 17. A later rise in RRA detectable EGF like activity was then observed over Days 18-20. The EGF activity from a Day 11 embryo chromatographed on high-performance liquid chromatography as a single peak, with very high recovery of activity, at a later elution position than mouse EGF or human EGF.     

Epidermal growth factor-like proteins in breast fluid and human milk

Epidermal growth factor (EGF), and the transforming growth factor-alpha (TGF-alpha) family of proteins, which also bind to the EGF receptor, have been associated with human breast cancer. The total EGF-like proteins were determined by a radioreceptor assay, and TGF-alpha by radioimmunoassay, in human milk and breast fluid samples. The breast fluids were collected by nipple aspiration from healthy premenopausal women. Both the 24 milks and 18 breast fluids assayed contained EGF-like proteins, at concentrations ranging from 32-600 ng/ml (median, 140 ng/ml), and 62-654 ng/ml (median, 205 ng/ml) respectively. Immunoreactive TGF-alpha proteins were detected at higher levels in 21 breast fluids (range, 0-50.0; median 5.1 ng/ml) than in 24 milk samples (range, 0-8.4; median, 0.8 ng/ml).     

A highly sensitive enzyme immunoassay (EIA) system for mouse epidermal growth factor (mEGF)

A highly sensitive two-site enzyme immunoassay system for mouse epidermal growth factor (mEGF) was developed, based on the sandwiching of an antigen between anti-mouse EGF IgG antibody-coated on a polystyrene bead and anti-mouse EGF Fab' antibody-linked peroxidase (horseradish peroxidase, EC. 1.11.1.7). The procedure is simple and rapid compared to a bioassay. Also, the Fab' antibody-peroxidase complex is more stable than the 125I-labeled antibody. Purified mEGF is detectable at a concentration as low as 3 pg/ml. The detection range was 0.3 to 680 pg/sample with 0.1 ml samples. Levels of immunoreactive mEGF in extracts from adult male mice well agreed with those determined by a radioimmunoassay and a radioreceptor assay. The submaxillary gland contained an extremely high concentration of EGF, while other tissues had low levels of EGF.     

Transforming growth factor type beta in normal human urine

TGF beta has been identified in normal human urine specimens from five individuals studied for five consecutive days. The peptide was extracted from urine using Sepralyte C1 beads. Detectable levels of [125I]TGF beta competing activity as measured by radioreceptor assay was found in about half of the specimens studied. The protein isolated from urine using C1 Sepralyte beads was further purified using Biogel P-60 column chromatography. Fractions were tested for TGF beta and EGF competing activity using radioreceptor assays. TGF beta and EGF extracted from urine are clearly separated by column chromatography. Two distinct EGF peaks and a single TGF beta peak were observed. Fractions having [125I]TGF beta competing activity were pooled and further purified using reverse-phase HPLC. HPLC fractions having [125I]TGF beta competing activity were tested for bioactivity using a soft agar assay. The fractions were capable of stimulating soft agar growth of AKR-2B (clone 84A) cells and cross reacted with a TGF beta antibody in a radioimmunoassay. The presence of TGF beta in normal human urine was also demonstrated by immunoblotting. These results also suggest that C1 bead extraction of urine specimens can be used as a rapid first step in purification of TGF beta.     

Solcoseryl in prevention of stress-induced gastric lesions and healing of chronic ulcers

Solcoseryl, a deproteinized extract of calf blood, protects the gastric mucosa against various topical irritants and enhances the healing of chronic gastric ulcerations but the mechanisms of these effects have been little studied. This study was designed to elucidate the active principle in Solcoseryl and to determine the role of prostaglandins (PG) and polyamines in the antiulcer properties of this agent. Using both, the radioimmunoassay and radioreceptor assay, EGF-like material was detected in Solcoseryl preparation. Solcoseryl given s.c. prevented the formation of stress-induced gastric lesions and this was accompanied by an increase in the generation of PGE2 in the gastric mucosa. Similar effects were obtained with EGF. Pretreatment with indomethacin, to suppress mucosal generation of prostaglandins (PG), greatly augmented stress-induced gastric ulcerations and antagonized the protection exerted by both Solcoseryl and EGF. Solcoseryl, like EGF, enhanced the healing of chronic gastro-duodenal ulcerations. This effect was abolished by the pretreatment with difluoromethylornithine, an inhibitor of ornithine decarboxylase, the key enzyme in the biosynthesis of polyamines. The healing effects of Solcoseryl and EGF was also reduced by prednisolone which decreased the angiogenesis in the granulation tissue in the ulcer area. These results indicate that Solcoseryl 1. contains EGF-like material, 2. displays the protective and ulcer healing effects similar to those of EGF and involving both PG and polyamines and 3. acts via similar mechanism as does EGF.     

Growth factor production by a human megakaryocytic tumor cell line

A recently described human megakaryocytic tumor cell line was analyzed for the presence of growth factor activity and was found to produce large quantities of transforming growth factor beta-like (TGF-beta) and basic fibroblast growth factor-like (bFGF) activities. Growth factor activities were identified using a radioreceptor assay for the TGF-beta-like activity, a heparin-binding assay for the b-FGF-like activity, and a demonstration of distinct biological activities for each type of factor. Tumor poly-A+ RNA revealed strong signals when probed with complementary DNA corresponding to bovine basic FGF and human TGF-beta and weak signals when probed with cDNA corresponding to epidermal growth factor (EGF) and TGF-alpha. The levels of EGF and TGF-alpha produced in the tumor line were too low to be detected by radioreceptor assays. Relative levels of messenger RNA encoding each of the growth factors reflected the relative levels of each of the respective factors tested. These data represent the first definitive identification of FGF-like activities in megakaryocytic-like cell lines. Interestingly, the line displayed little activity similar to platelet-derived growth factor (PDGF) when assayed either biochemically or by poly-A+ RNA analysis.     

Saliva from cystic fibrosis patients contains an unusual form of epidermal growth factor

Epidermal Growth Factor (EGF) was assayed in saliva collected from control subjects and cystic fibrosis (CF) patients, using both radioimmuno (RIA) and radioreceptor (RRA) assays. An intriguing finding was that the average ratio of the values found by RRA over those obtained by RIA was of 1.7 for normal subjects and of about 0.4 for CF patients. This observation could be understood following gel filtration analysis of EGF-like material in these salivary fluids. Whereas control saliva contained the expected 6 kDa EGF active peptide, the immunoreactive EGF material from CF patients eluted as a polydisperse macromolecular moiety. The poor biological reactivity of this material as assessed by radioreceptor assay suggests that this EGF anomaly may contribute to the physiopathology of cystic fibrosis, especially as the upper gastrointestinal tract differentiated functions may be the target of normal salivary EGF.     

Radioreceptor assay for epidermal growth factor.

An established cell line of human lung fibroblasts with a high number of surface receptorsfor mouse epidermal growth factor (mEGF) was used to develop a simple and highly sensitive radioreceptor assay for EGF. ¹²⁵I-Labeled mEGF competed mole for mole with unlabeled mEGF for specific receptors. Optimal range for discriminating EGF concentrations in body fluids and tissue extracts by a competitive binding assay was between 5 and 100 ng/ml. Interassay correlation of variation was 8.47% and the recovery of highly purified mEGF added to serum and urine samples was greater than 95%. Human serum and amniotic fluids contained about 24 and 4 ng/ml, respectively, of mEGF equivalents. Concentrations of mEGF in mouse urine and serum were highly variable and were 2- to 10-fold greater than that previously detected by radioimmune assay. Hypophysectomy nearly abolished submaxillary mEGF content in both male and female mice, but testosterone treatment of hypophysectomized animals restored normal concentrations of mEGF to the glands. mEGF added to culture medium disappeared with time as a function of the number of cellular EGF receptors indicating cellular degradation of the growth factor. The radioreceptor assay for EGF is based on the close biologic relationship between the cell receptor site and the native hormone and should prove to be a useful complementary tool to characterize the physiological role of EGF.     

In vivo evaluation of epidermal growth factor (EGF) receptor density on human tumor xenografts using radiolabeled EGF and anti-(EGF receptor) mAb 425

 In order to study the potential of non-invasive scintigraphic evaluation of the epidermal growth factor (EGF) receptor status in vivo, the biokinetics and tumor binding of ¹²⁵I-EGF and anti-(EGF receptor) mAb 425 were investigated in nude mice bearing human tumor xenografts with different EGF-receptor densities as determined by a radioreceptor assay. The results demonstrated a tumor uptake for both substances depending on the receptor level. The EGF receptor status, however, was reflected slightly better by the binding of EGF to tumor tissue compared to the mAb. The rapid blood clearance of EGF with a plasma half-life of less than 1 min led to a tumor-to-blood ratio of approximately 3 within 6 h after injection in tumors with a high receptor expression. A similar ratio for the mAb was not obtained before day 6 after injection. The absolute concentration of EGF, however, was low compared to the mAb. Therefore, it can be concluded that the EGF receptor status as a target for (radio)immunotherapy can be evaluated in vivo with EGF labeled with a short-life positron-emitting radionuclide or with monoclonal antibodies to the EGF receptor or their fragments. Received: 14 September 1995 / Accepted: 6 December 1995     

怀集石燕燕窝促细胞分裂活性的研究

1.借助Bio—Gel P—10柱层析法,由怀集石燕燕窝水提物部分纯化得一具有EGP活性的成分EGF-2。 2.在放射标记受体活性测定中,EGF-2与受体的竞争性结合曲线与小鼠EGF标准曲线相平行。EGF-2能显著刺激氚标记的胸腺嘧啶脱氧核苷对小鼠3T3成纤维细胞的掺入作用。这种活性不受抗小鼠EGF抗体的抑制。 3.借助Sephacry1-200 Superfine柱层析法,由上述水提物分离得一蛋白质组分(S-200Ⅰ+Ⅱ),对培养的人脐带淋巴细胞有促细胞分裂作用。并对培养的、经Con A转化的淋巴细胞有辅促细胞分裂作用。     

Radioimmunologic and radioreceptor studies of the pharmacokinetics of the enkephalin analog dalargin in man

Pharmacokinetics of dalargin, an opioid hexapeptide, was investigated on 7 males by two approaches. Dalargin radioimmunoassay was performed using a highly specific antiserum reacting only with the whole molecule. In radioreceptor assay lyophilized rat brain membranes containing opiate receptors were used. 2-6 min after intravenous introduction of 1-10 mg dalargin, immunoreactive dalargin blood concentration was lower than 0.5 ng/ml. The results of radioreceptor assay were presented as a biexponential curve with a fast main phase of activity changes (90%, characteristic time 1.5-5.0 min) and a slow "clearance" phase (10% of the substance, characteristic time 85-200 min). Prolonged presence of receptor-active substances in the blood can be attributed to the products of dalargin degeneration, namely its N-terminal penta- and tetrapeptides.     

Purification and partial characterization of a mitogenic protein released from preadipocytes of massively obese subjects

A protein released into the culture medium by omental preadipocytes of massively obese persons, which stimulates the replication of rat perirenal preadipocytes, has been purified to a high degree. By gel filtration chromatography, the molecular mass of the mitogenic protein was approximately 66,000 daltons (Da), while on sodium dodecyl sulfate - polyacrylamide gel electrophoresis, two subunits were obtained, relative masses (Mr) of approximately 31,000 and approximately 35,000. The isoelectric point of the approximately 66,000 Da entity was 5.6 +/- 0.2. By specific radioreceptor assay, the purified protein was related to epidermal growth factor and transforming growth factor alpha. It was not related to insulin-like growth factors I and II by radioimmunoassay and radioreceptor assay. We propose that the approximately 66,000 Mr protein, and other mitogenic proteins released by preadipocytes from massively obese persons, act through paracrine-autocrine mechanisms and may play a role in the development of the hyperplasia of enlarged fat cells characteristic of massive corpulence.     

Purification and characterization of epidermal growth factor (beta-urogastrone) and epidermal growth factor fragments from large volumes of human urine

We purified human epidermal growth factor (hEGF) and hEGF fragments from a benzoic acid precipitate of the materials not adsorbed to silica gel in 330 liters of human urine by a relatively brief, simple, and efficient method employing sequential batch absorption to and stepwise elution from CM-cellulose and DEAE-cellulose, Bio-Gel P-10 chromatography in 50 mM HCl, and three reverse-phase HPLC steps for final resolution and purification of hEGF components. Recovery of hEGF was 29%. Eight apparently homogeneous hEGF components were recovered, each of which had similar activities in a homologous hEGF radioimmunoassay and an EGF radioreceptor assay using human placental membranes. Amino acid composition analysis indicated that there were four pairs of components that represented intact 53-amino acid hEGF, hEGF-(1-52), hEGF-(1-51), and hEGF-(1-50); intact hEGF accounted for one-third of the total materials recovered. Automated Edman degradation of each component for at least 10 cycles revealed a single amino acid sequence identical to that proposed for human beta-urogastrone. Similar immunoreactive hEGF components were observed in similar proportions in freshly voided urine, indicating that they were not artifacts of the purification process. Thus, multiple forms of fully biologically active hEGF (i.e., beta-urogastrone) can be relatively easily and efficiently purified from large volumes of human urine.     

A biological assay for epidermal growth factor/urogastrone and related polypeptides

A bioassay for epidermal growth factor (EFG) which is rapid, specific, and extremely sensitive is described. As the bioassay detects as little as 25 pg of EGF, this assay is more sensitive than commonly used radioreceptor assays and nearly as sensitive as radioimmunoassays. This bioassay involves only the measurement of the proliferation of cultures of an EGF-requiring cell line and can be carried out in a quantitative manner over a 40-fold range of EGF concentrations.     

Synthesis of insulin-like growth factor I using N-methyl pyrrolidinone as the coupling solvent and trifluoromethane sulphonic acid cleavage from the resin

Insulin-like growth factor I (IGF-I), a protein of 70 amino acid residues and 3 cystine bridges, has been synthesized by two solid phase Boc methods. The first method used N-methylpyrrolidinone as the solvent with single coupling cycles while the second synthesis used dimethylformamide and dichloromethane as the solvents with a double-coupling protocol. In both cases, trifluoroacetic acid/trifluoromethanesulphonic acid cleavage of the peptide from the resin was employed. Purification of the cleavage products followed by removal of the S-acetamidomethyl protecting groups gave reduced peptides which were then oxidized under conditions favouring the formation of the correct disulphide bonds. The purified synthetic IGF-I peptides were full agonists of natural IGF-I in a radioimmunoassay, in an IGF-I radioreceptor assay, in a bioassay which measures the stimulation of protein synthesis in rat L6 myoblasts and in an IGF-binding protein competitive binding assay. Moreover, in each of these assays, the synthetic IGF peptides were found to be at least 70% as potent as natural IGF-I.     

Chemical synthesis of bovine transforming growth factor-alpha: synthesis, characterization and biological activity

Bovine transforming growth factor-alpha (bTGF-alpha) is a 50 amino acid polypeptide with three disulfide linkages. In order to evaluate the biological function of this peptide, bTGF-alpha was synthesized via an automatic synthesizer and purified to homogeneity in high yield. The integrity of this synthetic peptide was confirmed by chemical analyses and bioassays. In a bovine liver radioreceptor assay, bTGF-alpha competes with radiolabeled EGF and has activity comparable to mEGF and hTGF-alpha. Compared to hEGF, bTGF-alpha elicits a greater response in a bovine mammary cell proliferation.     

Culture of transformed horseradish roots as a source of fusicoccin-like ligands

Endogenous fusicoccin (FC) or related substances were sought in horseradish (Armoracia rusticana P.) roots. An actively growing root culture was derived from plants transformed with Agrobacterium rhizogenes. The presence of FC-like substances in ethanolic extracts from roots was established in a radioreceptor binding assay with plasmalemmal FC receptors and in radioimmune analysis with an antiserum specific for FC A. FC-like ligands were found in the tissue and medium of aseptically grown culture.Abbreviations FC fusicoccin - GC/MS gas chromatography/mass spectrometry - RIA radioimmunoassay - RRA radioreceptor analysis - BSA bovine serum albumin - Mes 4-morpholineethanesulfonic acid - HPLC high performance liquid chromatography     

Elevated epidermal growth factor receptor binding in plutonium-induced lung tumors from dogs

The objective of this study is to examine and characterize epidermal growth factor receptor (EGF-R) binding in inhaled plutonium-induced canine lung-tumor tissue and to compare it with that in normal canine lung tissue. Crude membrane preparations from normal and lung-tumor tissue from beagle dogs were examined in a radioreceptor assay, using 125I-labeled epidermal growth factor (EGF) as a ligand. Specific EGF receptor binding was determined in the presence of excess unlabeled EGF. We have examined EGF receptor binding in eight lung-tumor samples obtained from six dogs. Epidermal growth factor receptor binding was significantly greater in lung-tumor samples (31.38%) compared with that in normal lung tissue (3.76%). Scatchard plot analysis from the displacement assay revealed that there was no statistical difference in the binding affinity but significantly higher concentration of EGF-R sites in the lung-tumor tissue (619 fmol/mg) than in normal lung tissue (53 fmol/mg). The increase in EGF-R number in plutonium-induced dog lung tumors does not seem to correlate with increase in the initial lung burden exposure to plutonium. Our results demonstrate that there is a significant increase in EGF-R binding in inhaled plutonium-induced dog lung tumors.     

Isolation and partial characterization of canine epidermal growth factor/urogastrone.

Canine epidermal growth factor (EGF)/urogastrone was partially purified from dog urine by fractional precipitation with (NH4)2SO4, ion-exchange chromatography with DEAE-cellulose DE-52, gel filtration with Sephadex G-50, and a second DE-52 chromatography, to yield receptor-competing activity equivalent to 13 micrograms of standard mouse EGF/litre of starting urine. The purification was monitored by a competitive radioreceptor assay using fixed monolayers of A431 cells. The partially purified canine EGF/urogastrone demonstrated a growth-stimulating activity in 3T3 mouse fibroblast cells as potent as mouse EGF. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one major peptide component with an Mr similar to that of mouse EGF, and two minor peptides of slightly higher Mr. The major peptide component was isolated after reduction and its amino acid composition was determined.