Naomi Minami

Expression of Epidermal Growth Factor Receptor in Fetal Mouse Submandibular Gland Detected by a Biotinyltyramide-based Catalyzed Signal Amplification Method

Branching morphogenesis of the fetal mouse submandibular gland (SMG) can be modulated in vitro by stimulation or inhibition of the epidermal growth factor receptor (EGFR). Because the mRNAs for EGF and EGFR are detectable in RNA of SMG rudiments isolated directly from fetuses, the EGF system probably operates physiologically as a regulator of SMG morphogenesis. However, neither EGFR protein nor its precise cellular localization has been characterized in the fetal SMG. Here we show EGFR protein in fetal mouse SMG by immunoprecipitation, affinity labeling, ligandinduced autophosphorylation, and immunohistochemistry. SMGs from E16 fetuses (day of vaginal plug = EO) were labeled with [35S]-cysteine/methionine and homogenized. After addition of specific antibody to EGFR, the immunoprecipitate was isolated, resolved by polyacrylamide gel electrophoresis, and detected by autoradiography. A single band of 170 kD was detected, corresponding to the EGFR protein. Affinity labeling with [125I]-EGF of the membrane fraction of E18 SMG also revealed a prominent band at 170 kD, showing that this EGFR protein can bind specifically to its ligand. Incubation of SMG membranes from E18 fetuses with EGF in the presence of [γ-32P]-ATP, followed by immunoprecipitation with anti-phosphotyrosine antibody also showed a single band at 170 kD, demonstrating autophosphorylation of the EGFR in response to binding of its ligand. Immunohistochemical localization of the cellular sites of EGFR in the fetal SMG required use of a catalyzed signal amplification procedure, with biotinyltyramide as the amplifying agent. EGFR was localized predominantly, if not exclusively, in cell membranes of epithelial cells of the rudiment, whereas staining of mesenchymal cells was equivocal. Staining was strongest on duct cells, and weak on cells of the end-pieces. These findings clearly show that a functional EGFR protein is expressed in fetal SMG chiefly, if not exclusively, on epithelial cells.

Influence of age on epidermal growth factor receptor level in the rat brain

The influence of age on125I-epidermal growth factor (EGF) binding to rat brain plasma membranes was investigated. The specific binding of EGF to membranes decreased gradually with age in both male and female rats. There was no significant difference in the specific binding between males and females. Scatchard analysis of the binding data showed that the decrease in EGF binding with age was due to a decrease in the number of EGF receptors.

Effect of streptozotocin-induced diabetes on epidermal growth factor receptors in rat liver plasma membrane.

The effect of streptozotocin-induced diabetes on 125I-labeled epidermal growth factor (EGF) binding was studied in microsomal membranes from rat liver. The binding of EGF in membranes from diabetic animals was significantly low, the value being about 60% of the control level. Scatchard analysis of the binding data clearly showed that the decrease in EGF binding was due to a decrease in the number of receptors. Treatment of diabetic animals with insulin restored EGF receptors to control levels, whereas the treatment with triiodothyronine had no effect. Serum EGF concentrations measured were almost the same among the control, diabetic, and insulin-treated diabetic groups. These results suggest that insulin deficiency in vivo causes a decrease in hepatic EGF receptors.

Male mouse submaxillary gland secretes highly toxic proteins

Submaxillary gland saliva induced by phenylephrine from male mice was highly toxic to guinea-pigs, rats and hamsters, whereas the toxicity was relatively low to mice. One of the toxic components in the saliva was isolated as a kallikrein-like enzyme.

Influences of the submaxillary gland of male mice on the immune response to sheep red blood cells.

Removal of the submaxillary gland (SMG) from male but not female mice caused a suppressed immune response to sheep red blood cells. Administration of a SMG saline extract from male mice to SMG-ectomized males restored the suppressed response to control levels. This suggests that the male mouse SMG contains a factor(s), possibly of an endocrine nature, capable of influencing cells involved in an immune response.

Genetic variation in esteroproteases in the mouse submandibular gland.

9 isozymes of esteroproteases were detected by column isoelectric focusing of submandibular gland extracts from four inbred strains of male mice. A marked strain variance in the esteroprotease isozymes was found among the strains.

Effect of Autonomic Agents on the Secretion of N-Acetyl-β-Glucosaminidase of Mouse Submaxillary Gland

The secretion of N-acetyl-β-glucosaminidase of mouse submaxillary gland into saliva was stimulated by norepinephrine and phenylephrine but not by pilocarpine and isoproterenol. The stimulative effects of the α-adrenergic agents were inhibited by α-blockers, phentolamine, and phenoxybenzamine. These results suggest that the secretion of the enzyme is regulated through α-adrenergic receptors.

An enzyme immunoassay for mouse epidermal growth factor utilizing a liquid phase double-antibody system

An enzyme immunoassay for mouse epidermal growth factor (EGF) involving a liquid phase double-antibody system was developed. The EGF-β-galactosidase conjugate prepared was stable for at least 8 months. By this method, EGF was detectable at a concentration as low as 20 pg per tube. The concentrations of EGF in various tissues of mice are also presented.

Purification and tissue distribution of rat epidermal growth factor.

1. Two forms of rat epidermal growth factor, EGF-I and EGF-II, were purified to homogeneity from male rat submandibular glands. 2. The mol. wts of EGF-I and -II were estimated to be 5200 and 5400, respectively, both of them having an apparent biological activity. 3. The antiserum against EGF-II strongly cross-reacted with EGF-I; however, it did so only slightly with mouse or human EGF. 4. EGF was detected by radioimmunoassay in various tissues of male and female rats, and the concentrations of rat EGF in the submandibular gland, parotid gland, sublingual gland, and liver were significantly higher in the male than in the female.

Biochemical properties of epidermal growth factor in the mouse kidney

Epidermal growth factor (EGF) concentration in the mouse kidney was exceedingly low when compared with the submandibular gland level. Gel filtration of kidney extract showed that kidney EGF had the same molecular weight as the submandibular gland peptide. The isoelectric point of kidney EGF was between pH 4.3 and 4.6. From reversed phase HPLC, two species of EGF, alpha-EGF and beta-EGF, were clearly detected in the kidney sample.