Masanori Kashimata

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.

Intracellular signalling cascades activated by the EGF receptor and/or by integrins, with potential relevance for branching morphogenesis of the fetal mouse submandibular gland.

The fetal mouse submandibular gland grown in vitro is a valuable model system to study epithelio-mesenchymal interactions vital for branching morphogenesis. This basic developmental process is dependent upon simultaneous engagement of receptors for growth factors and components of the extracellular matrix. Here we review intracellular signalling cascades mediated by activation of the EGFR in the developing mouse submandibular gland.

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.

Immunohistochemical analysis of EGF in epiphyseal growth plate from normal, hypophysectomized, and growth hormone-treated hypophysectomized rats.

Epiphyseal growth plate cartilages from the proximal tibia of normal, hypophysectomized, and growth hormone (GH)-treated hypophysectomized rats were subjected to immunohistochemistry for detection of epidermal growth factor (EGF). In the normal growth plate, EGF was distributed mainly in the proliferative zone. Hypophysectomy resulted in considerable atrophy of the chondrocytes and the cartilage matrix (a decreased number of mature-type chondrocytes and a decreased ratio of proliferating to hypertrophic chondrocytes) and a significant diminution of EGF immunoreactivity. Treatment with GH reversed these effects of hypophysectomy, causing an increased thickness of the growth plate and EGF-reactive sites in all chondrocyte layers. The most intense immunostaining for EGF, however, was frequently seen in the nuclei of chondrocytes with flattened appearance. It appears that EGF could be incorporated or synthesized in chondrocytes having marked mitogenic activity. The present results, taken with previous data on EGF involvement in growth of cartilaginous tissue in vivo and in vitro, strongly suggest that EGF-immunoreactive chondrocytes are involved in cartilage proliferation and growth under the specific influence of GH.

Peroxidase activity in salivary glands and saliva of beige (Chediak-Higashi) mice

Peroxidase activity was lower in the submandibular and parotid glands of beige mice than in controls. A lower level of activity was also observed in the isoproterenol-elicited saliva of beige mice.

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.

Sialidase in rat salivary glands. Characteristics of soluble sialidase in salivary glands and other tissues

In this study, we elucidated some characteristics of soluble sialidases in the rat salivary glands, brain, liver and kidney. 1) Soluble sialidases in the submandibular and parotid glands were inactivated by about 75 and 60%, respectively, and the enzymes in the brain, liver and kidney were also inactivated by 16-48%, when preincubated at 40 degrees C for 1h. When preincubated at 60 degrees C, the enzymes in all tissues were completely inactivated within 10 min. 2) Ca2+ ion in low concentrations tended to activate soluble sialidases in both the submandibular and parotid glands, but had no apparent effect on the enzymes in the brain, liver and kidney. The enzyme in the submandibular gland tended to be activated also by low concentrations of Mg2+. Both Cu2+ and Hg2+ ions caused a marked inhibition on the soluble sialidases in all tissues. 3) The molecular weight of soluble sialidases in the submandibular and parotid glands was determined to be about 68,000 and 46,000, respectively, by means of Sephadex G-200 gel filtration. The molecular weight of the enzymes in the brain and liver was almost similar to that of the enzyme in the parotid gland. In the kidney, soluble sialidase was separated into two enzymes, one having high (more than 500,000) and the other having low (about 46,000) molecular weight. 4) By isoelectric focusing analysis, three isozymes were detected in the submandibular gland and brain sialidases and two isozymes detected in the enzymes of other tissues. The isoelectric point of the major isozymes in the submandibular and parotid glands was 6.4 and 6.9 respectively. In the brain, liver and kidney sialidases, the isozymes with isoelectric point in the range of 4.4 to 6.7 were detected.