Biomedical subjects
Publications and source records attributed to E Nexø.
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Epidermal growth factor (EGF) is mainly produced in the submandibular glands (SMG) and in the kidneys. It has recently been reported that EGF-related ligands may induce their own biosynthesis (autoinduction) in vitro. In the present paper, we investigated whether chronic systemic treatment with EGF influenced the amount of endogenous EGF in the SMG and kidneys. Eight-week-old female Wistar rats were treated with subcutaneous injections of placebo (n = 16) or human recombinant EGF (150 micrograms/kg per day, n = 8) for 4 weeks. Urine was sampled the last 24 h of the study period. At the time of killing, the SMG and the kidneys were removed. The SMG was larger in the EGF-treated animals, 229.8 +/- 35.5 (mean +/- SD) mg than in the control animals, 181.7 +/- 18.1 mg (P < 0.01). The total EGF content was smaller (0.51 +/- 0.15 vs. 1.12 +/- 0.40 nmol EGF/SMG, P < 0.001). The kidneys were larger in the EGF-treated animals (1.38 +/- 0.08 vs. 1.28 +/- 0.08 g, P < 0.05), but the EGF content and urinary excretions were not changed. In conclusion, chronic systemic treatment with EGF causes growth of the SMG with concomitantly reduced contents of EGF, and growth of the kidneys with unchanged content and excretion of EGF. These findings suggest that EGF may play a part in the regulation of the growth of the SMG and in EGF biosynthesis.
Kidney cortex is a main target for circulating vitamin B12 (cobalamin) in complex with transcobalamin (TC). Ligand blotting of rabbit kidney cortex with rabbit 125I-TC-B12 and human TC-57Co-B12 revealed an exclusive binding to megalin, a 600-kDa endocytic receptor present in renal proximal tubule epithelium and other absorptive epithelia. The binding was Ca2+ dependent and inhibited by receptor-associated protein (RAP). Surface plasmon resonance analysis demonstrated a high-affinity interaction between purified rabbit megalin and rabbit TC-B12 but no measurable affinity of the vitamin complex for the homologous alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor related protein (LRP). 125I-TC-B12 was efficiently endocytosed in a RAP-inhibitable manner in megalin-expressing rat yolk sac carcinoma cells and in vivo microperfused rat proximal tubules. The radioactivity in the tubules localized to the endocytic compartments and a similar apical distribution in the proximal tubules was demonstrated after intravenous injection of 125I-TC-B12. The TC-B12 binding sites in the proximal tubule epithelium colocalized with megalin as shown by ligand binding to cryosections of rat kidney cortex, and the binding was inhibited by anti-megalin polyclonal antibody, EDTA, and RAP. These data show a novel nutritional dimension of megalin as a receptor involved in the cellular uptake of vitamin B12. The expression of megalin in absorptive epithelia in the kidney and other tissues including yolk sac and placenta suggests a role of the receptor in vitamin B12 homeostasis and fetal vitamin B12 supply.
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The concentration of endogenous cobalamin (Cbl) in cow milk was 3.3 nM while the Cbl-binding capacity was 0.05 nM. Both endogenous and newly added Cbl showed similar quantitative distribution between a 280 kDa protein complex (45%) and a 43 kDa Cbl-binder (55%). Long time incubation, as well as urea treatment, was accompanied by a slow release of the 43 kDa Cbl-binder from the 280 kDa fraction. No other Cbl-binding proteins appeared after these procedures. The 43 kDa binder from cow milk, depleted of the ligand by urea treatment, reacted with Cbl even in the presence of a B12-analogue cobinamide (Cbi) at the ratio Cbl:Cbi = 1:40. The stokes radius of the binder changed from 2.7 nm for the Cbl-free protein to 2.5 nm for the Cbl-saturated form and the Cbl-saturated binder was able to displace human transcobalamin (TC) from the TC-receptor. The interaction between the protein and Cbl was significantly suppressed at pH 2.0. The N-terminal sequence of the purified 43 kDa Cbl-binder revealed homology with TC from human and rabbit plasma. In conclusion we have shown that TC is the main Cbl-binding protein in cow milk. This is surprising, since previous studies on human and rat milk have shown another Cbl-binder, apo-haptocorrin, to be the dominating Cbl-binding protein.
Systemic treatment with epidermal growth factor (EGF) induces growth of all wall layers in the urinary tract of pigs and rats. The present study was initiated to describe morphological and biochemical changes in the bladder smooth muscle from rats treated with EGF for 4 weeks. Eight-week-old female Wistar rats were treated with subcutaneous injections of vehicle (n = 16) or EGF (n = 8, 150 micrograms/kg per day) for 4 weeks. After EGF treatment the bladders were increased in weight [74.4 +/- 0.4 vs 122.1 +/- 0.5 mg, P < 0.001 (mean +/- SEM)]. Sodium dedecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analyses of six bladders from each group revealed that the total amounts of actin, myosin and desmin were statistically significantly increased by 62%, 61% and 154%, respectively. The relative amounts of actin and myosin were unchanged whereas the desmin to actin ratio was significantly increased - as previously described in rat bladder smooth muscle hypertrophy. Light and electron microscopy of two bladders from each group revealed increased wall thickness involving all wall layers. The smooth muscle fibres at a midventral bladder location seemed only slightly hypertrophic-some degree of hyperplasia was therefore suspected. In conclusion, EGF treatment for 4 weeks induced a net synthesis of contractile and cytoskeletal proteins in the urinary bladder smooth muscle.
Twenty-four male Wistar rats, 8 weeks old, were allocated into three groups and treated with human recombinant epidermal growth factor (EGF) administered subcutaneously in doses of 0, 30, and 150 micrograms/kg per day for 4 weeks. Blood sampling was done every 2nd week and urine sampling was done for 2 consecutive days every week. The most striking finding was that the ureters were dose dependently enlarged, due to growth of all layers of the ureteric wall. The urothelium of the bladder showed considerable hyperplasticity with a widening of the basal proliferative compartment and a normal differentiation pattern as observed by the expression of carbohydrate epitopes, characterized with lectinohistochemistry. Blood examination revealed a decrease in blood haemoglobin concentration and a slight increase in serum creatinine concentration in the high-dose group. There were no effects of EGF on the urinary excretion of electrolytes, proteins, and endogenous EGF.
Epidermal growth factor (EGF) is considered to be important in mammalian neonatal growth and development. In order to clarify its developmental role, we have investigated, by immunohistochemistry, the localization of EGF and the time of its first appearance in various organs from a series of 25 midtrimester human fetuses with a gestational age ranging from 13 to 22 weeks. The first detectable EGF immunoreactivity occurred in week 15-16 fetuses in the placenta, the skin, the distal tubules of the kidney, the surface epithelium of the stomach, and the tips of the small intestinal villi, as well as in a few Paneth cells. Glandular structures, such as the glands of the cardia and the pyloric part of the stomach, Brunner's glands of the duodenum, the pancreas, and the submucous glands of the trachea, showed positive EGF immunoreactivity later (week 17). Thus, apart from the kidney, staining of the surface epithelia seems to precede staining of the EGF-producing glandular structures and EGF is not present in the glands before these have already differentiated.
Different molecular weight forms of epidermal growth factor (EGF) are present in vivo and this makes quantitation of EGF difficult. Most immunoassays employ antibodies against 6-kDa EGF, and such assays are likely to underestimate the amount of high molecular weight forms of EGF. The purpose of the present study was to develop a processing-independent ELISA which is able to quantitate different molecular forms of rat EGF with equimolar potency. Our "old ELISA" used two polyclonal antibodies against rat submandibular gland EGF as catching and detecting antibodies, and 6-kDa EGF purified from rat urine as calibrator. This assay was modified to a processing-independent ELISA by converting the different forms of EGF in the samples as well as the calibrator to the same immunoreactive form of EGF prior to analysis. This could be achieved by trypsinization because trypsin cleaved the different molecular forms of rat urinary EGF to a single immunoreactive form. We applied both the "old ELISA" and the processing-independent ELISA on different molecular forms of EGF and demonstrated that the "old ELISA" underestimated high molecular weight forms by two thirds. The relative amounts of high and low molecular weight forms of EGF in urine have been debated, since different results have been obtained by different techniques. In order to address the problem it is important to quantitate the different molecular forms with equimolar potency. Employing the processing-independent ELISA we find that high molecular weight forms of EGF constitute 40% and 6-kDa EGF 60% of EGF in fresh rat urine.
Blood samples were drawn every second hour during a 24-h period from healthy elderly subjects. Plasma levels of cobalamin, albumin, transcobalamin-bound cobalamin and unsaturated binding capacity of transcobalamin and haptocorrin were determined. During night, all components decreased in parallel with plasma albumin. The nocturnal decreases were therefore ascribed to plasma volume changes due to bed-rest. Reports on the recently proposed role of cobalamin for the biological clock were reviewed, but the proposed role could not be explained by this study.
We have studied the interaction between transcobalamin (TC) and the ligands cobalamin (Cbl) and cobinamide (Cbi). Partially purified TC from bovine milk was depleted of endogenous Cbl by 8 M urea treatment. Unsaturated TC was adsorbed on CM-Sepharose in order to ensure fast separation of the matrix-bound protein from the reaction medium. The forward reaction TC+Cbl-->TC-Cbl (rate constant k+Cbl) and the backward reaction TC-Cbl-->TC+Cbl (k-Cbl) were followed in time. A single-step binding model (with no intermediate protein-ligand complex) was sufficient to fit the data. The calculated rate constants were k+Cbl = 0.6 nM-1 min-1 and k-Cbl = 1.3 x 10(-4) min-1, which corresponded to the TC-Cbl dissociation constant KDCbl = 0.2 pM. Reaction between TC and Cbl developed against electrostatic forces, and the effective charges of the interacting species were estimated as both +1 or both -1. The competition between Cbl and Cbi for TC was studied, which resulted in determination of the relevant rate constants for Cbi: k+Cbi = 0.03 nM-1 min-1, k-Cbi = 0.03 min-1, and KDCbi = 1 nM. Slow dissociation of TC-Cbl guarantees its stability in plasma for 5-10 h, while Cbi bound to TC would be transferred to haptocorrin in less than 1 h.
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The EGF receptor is a transmembrane glycoprotein exerting mitogenic effects on epithelial cells. The purpose of the present study was to develop a sensitive enzyme-linked immunosorbent assay (ELISA) for determination of the epidermal growth factor receptor (EGFR) protein to examine whether the receptor was overexpressed in head and neck squamous cell carcinomas compared with the normal counterpart, and to establish whether clinicopathological correlations were present by investigating a broad spectrum of parameters (tumour size, clinical stage, positive lymph nodes, tumour site, histological grade, keratinisation, preoperative irradiation and clinical outcome). The assay employs two commercially available monoclonal antibodies, both detecting protein epitopes. The material comprises 60 head and neck carcinomas, corresponding normal tissue and normal oral mucosa from healthy individuals. The study demonstrates significantly higher receptor levels in tumours compared with normal tissue (P < 0.002) and a range in tumours and normal tissues of 0.4-10.5 and 0.1-4.3 nmol g-1 membrane protein respectively. Quantitation of receptors in normal mucosa emphasises the importance of using the patients' corresponding normal tissue, because using the patients' mucosa resulted in 83% overexpression, while using normal mucosa from healthy individuals only demonstrated overexpression in 50% of cases. No significant clinicopathological correlations could be established, although the mean values for EGFR increased with tumour size and advanced clinical stage. Furthermore, the prognostic value concerning disease-free survival, recurrence and the time interval for recurrence were investigated but no significance could be demonstrated. In conclusion, the investigation supports the theory of overexpression of EGFR protein as a common motif for malignant epithelial tumours, but limitations in interpretations are demonstrated and discussed further.
Epidermal growth factor (EGF) is a growth-promoting peptide that is synthesized in the distal tubules of the kidney and excreted in urine. EGF has been suggested to play a role in the repair after renal tissue damage, as well as in compensatory growth of the remaining kidney after uninephrectomy. The present study examined the urinary EGF excretion after uninephrectomy and transplantation among relatives. The urinary EGF excretion rate and the glomerular filtration rate (GFR) were followed for 26-54 days in 16 healthy kidney donors and nine recipients. After uninephrectomy the median urinary EGF excretion rate in the donors was not 50% of the pre-operative value, but around 65% (95% confidence limits of the median on the fifth post-operative day: 59-72%). This suggests that there is a compensatory increase in the EGF excretion rate from the remaining kidney of around 30% after uninephrectomy. A similar compensatory increase was demonstrated for GFR, indicating that the compensatory changes in EGF excretion rate and GFR might be correlated. In the transplanted kidneys, GFR was consistently around 15% lower and EGF excretion rate around 40% lower than in the corresponding kidneys remaining in the donors. This might reflect ischaemic and drug-induced damage of the transplanted kidneys. The present study demonstrated a compensatory increase of around 30% in urinary EGF excretion from the remaining kidney after uninephrectomy in healthy humans. Whether EGF plays a role in the adaptive processes in the remaining kidney or whether changes in EGF excretion are merely of a secondary nature is still uncertain.
Plasma gastrin concentration increases in late gestation and reaches a peak at birth or shortly after birth in many species ('neonatal hypergastrinaemia'). We investigated the hypothesis that gastrin and gastric acid secretion in the neonate is influenced by the final rise in plasma cortisol associated with spontaneous (vaginal) birth. Caesarean-delivered (CD, n = 28) or vaginally delivered (VD, n = 24) premature or full-term piglets (97-115 days gestation) were killed immediately after birth (using pentobarbitone). Compared with newborn CD pigs, the newborn VD pigs had significantly higher (P < 0.05) concentrations of cortisol (669 +/- 60 versus 223 +/- 2 nM) and gastrin (57 +/- 4 versus 33 +/- 1 pM) in plasma, and significantly lower gastric fluid pH (3.3 +/- 0.2 versus 4.7 +/- 0.3), amidated (bioactive) gastrin in the antrum (550 +/- 77 versus 1220 +/- 29 pmol g-1) and glycine-extended (precursor) gastrin in the antrum (81 +/- 10 versus 143 +/- 5 pmol g-1). There were significant linear correlations between log plasma cortisol values and plasma gastrin (r = 0.40, P < 0.05) or gastric fluid pH (r = -0.51, P < 0.05) in newborn pigs. The effects of cortisol in the immediate postnatal period were investigated in forty-one CD pigs born at 111-112 days gestation and treated with saline, metyrapone (an inhibitor of cortisol synthesis) or adrenocorticotrophic hormone (ACTH) from 0 to 7 days after birth. At 7 days, plasma gastrin in ACTH-treated pigs (elevated plasma cortisol) was significantly lower than in saline-treated pigs, but not different from that in metyrapone-treated pigs (low plasma cortisol). No treatment effects were observed postnatally for antral gastrin and fundic cobalamin-binding protein concentrations. These results suggest that in the intrapartum period the high circulating cortisol levels stimulate the normal rise in gastrin and acid secretion associated with spontaneous vaginal delivery in the piglet, whereas postnatally, cortisol is unlikely to play an important role in the subsequent development of gastrin and acid secretion.
Epidermal growth factor (EGF) in pharmacologic doses is able to induce growth and development in the fetus and the newborn. To investigate the opposite situation, the effects of insufficient amounts of EGF during development, we wanted to establish an in vivo model with a state of EGF deficiency. This was attempted by induction of autoimmunity to EGF in rats. Twenty rats were immunized with EGF. Fifteen of these developed autoantibodies against EGF, which, as judged by Scatchard analysis, had a median apparent affinity constant of 14 x 10(9) L/mol and a median concentration of binding sites of 20 x 10(-9) mol/L. The antibodies recognized purified EGF from the submandibular glands (6 kD) and from urine (45 kD) and further native EGF in saliva and urine. The cross-reactivity toward transforming growth factor-alpha was below 3%. Binding of EGF by antibodies inhibited its binding to the EGF-receptor by approximately 97% in vitro. Investigation of in vivo metabolism of antibody-bound 125I-EGF confirmed these results, that is, the antibodies were able to inactivate EGF. The adult rats were unaffected by the induction and presence of autoantibodies, and the EGF-containing organs did not show any histologic signs of inflammation or tissue damage. Furthermore, as judged by immunohistochemistry, no major changes in the distribution and tissue concentration of EGF were seen in the adult rat. These results show that it is possible to induce homologous antibodies that can inhibit the binding of EGF to its receptor and further suggest that circulatory EGF is of no physiologic importance in the healthy, adult rat.
We have used rats with epidermal growth factor (EGF) autoantibodies to study the role of EGF deficiency during perinatal development. The study was focused on organs known to contain EGF or its receptor. Compared with controls, the offspring of autoimmune rats had a higher perinatal mortality and a lower birth weight. The weight of the lungs was particularly low in the offspring of EGF-immunized rats, and morphologically the lungs from the surviving pups seemed atelectatic and had alveolar duct dilatation, which indicates mild respiratory distress syndrome. Judged from immunohistochemical studies, the amount of surfactant protein-A was decreased, suggesting a delayed lung maturation. The offspring of EGF-immunized rats had dry and wrinkled skin. The skin was thin and the hair follicles were immature. This suggests a role for EGF in the growth and development of the skin. The liver/body weight ratio was lower in pups from EGF-immunized rats. This difference was, however, not significant (p = 0.07), but flow cytometric analyses showed a significantly lower proportion of the liver cells from newborn EGF-deficient pups to be in S-phase and indicated that these cells were larger than liver cells from controls. To study possible alterations in EGF binding, 125I-EGF was injected i.v. in newborn rats. 125I-EGF bound in all the organs investigated. The binding is listed in decreasing order: liver, gut, skin, kidney, and lungs. In the pups from EGF-immunized rats, the lungs and the skin bound a significantly higher amount than the controls.(ABSTRACT TRUNCATED AT 250 WORDS)