Murine Submaxillary Glands Research Articles

A new assay for nerve fiber repulsion

In inflammatory lesions, sympathetic nerve fibers get lost soon after the start of inflammation. To be able to identify and examine the factors that are responsible for this repulsion of the sympathetic nerve fibers we established an in vitro assay. Sympathetic trunk ganglia of postnatal mice were used for the outgrowth of axons. They were plated on poly-D-lysine-coated culture slides. Axons were encouraged to grow out by the addition of nerve growth factor-7S (NGF) from murine submaxillary gland. Using live imaging it was clearly shown that SEMA3F is a nerve-repellent factor. Two different types of nerve fiber repulsion were observed. The assay turned out to be an excellent model for the investigation of axon guidance factors of sympathetic neurons (nerve fiber repulsion/nerve fiber attraction).

Radioprotection of the murine submandibular gland by isoproterenol: autoradiography study with 3H-leucine

Irradiation of the salivary glands results in the generation of free radicals from metal ions present in the secretory granules of acinar cells, a process that is believed to exacerbate radiation damage to the salivary glands. We therefore conducted a comparative investigation of radiation damage to the acinar cells of murine submaxillary glands in which granule secretion had been induced, and used autoradiography to visualize the pathological changes. Male BALB/c mice, at 8 weeks of age, were divided into four groups: a no-isoproterenol (IPR) and no-irradiation group (group I), a no-IPR, irradiated group (group II), an IPR, no-irradiation group (group III), and an IPR, irradiated group (group IV). Intraperitoneal injections of IPR were used, and 3 h later, the submaxillary region was irradiated with X-rays at a dose of 10 Gy. Three days after the irradiation, 3H-leucine was administered, and submaxillary glands were removed at predetermined times. Thin sections were prepared, and light- and electron-microscope autoradiography was performed. The number of reduced silver particles per unit acinar cell area was determined by light-microscopic autoradiography, and the proportion of reduced silver particles in the rough endoplasmic reticulum, the Golgi apparatus, and secretion granules was determined by electron-microscopic autoradiography. The result indicated that the effects of the radiation on the secretory potential of the submaxillary glands were diminished in acinar cells with a higher secretory granule content.

Human basophilic cell differentiation promoted by 2.5S nerve growth factor.

In liquid cultures of human cord blood mononuclear cells, the activities of the 2.5S nerve growth factor (NGF) inducing basophil and eosinophil differentiation were investigated. Various concentrations of immunopurified 2.5S NGF derived from murine submaxillary glands were added to cultures with or without conditioned medium from a human T cell line (Mo-CM), which has previously been shown to produce activities stimulating granulocyte-macrophage colonies. Addition of NGF led to significant increases in differentiation of basophilic cells accompanied by histamine synthesis at 2 weeks in vitro; eosinophil differentiation was not increased in these cultures. In addition, NGF could be shown to amplify basophil differentiation induced by Mo-CM, and the activity of NGF inducing basophil differentiation was dependent on the presence of T lymphocytes. These results indicate that NGF stimulates T-lymphocyte-dependent basophilic cell differentiation from human cord blood progenitors and may in this way support differentiation of basophils or mast cells in vivo at sites of allergic tissue inflammation.

Isolation of multiple biologically and chemically diverse species of epidermal growth factor

We have analyzed several lots of epidermal growth factor (EGF) purified from murine submaxillary glands including “receptor grade” EGF from Collaborative Research and EGF from Boehringer Mannheim Biochemicals. New England Nuclear uses “receptor grade” EGF to produce 125I-labeled EGF. Though these reagents are reported to be homogeneous, we found them to be a mixture of six species. A method was developed to separate this mixture into its component parts. The individual components were chemically characterized and tested for biological potency. N-terminal sequence analysis of the unfractionated EGF-mixture reveals three different sequences starting with residues 1, 2, or 3 of the mature peptide. Each component exhibited different degrees of mitogenic and EGF receptor binding activity indicating that the N-terminal region contributes to the biological response. The species representing the complete EGF peptide is the most active species in all biological assays. A rapid method for purification of homogeneous complete EGF from commercial EGF preparations is described.

Growth factor-binding proteases in the murine submaxillary gland: isolation of a cDNA clone.

The submaxillary gland of the adult male mouse contains a number of serine proteases, several of which are involved in the proteolytic processing of precursors to growth factors and other biologically active polypeptides. Here we report the isolation and identification of a cDNA clone corresponding to one of the proteases, the type B of the epidermal growth factor-binding protein. A pronounced sequence homology was found between the predicted activation peptide of this protease and the NH2-terminal extension of the nerve growth factor alpha subunit, suggesting that the latter protein has an uncleaved activation peptide attached to its NH2 terminus.

Effects of androgens on the activity of steroid-metabolizing enzymes in the murine submaxillary gland.

Effects of androgens on the activity of androgen-metabolizing enzymes have been investigated in the murine submaxillary gland. 5 alpha-reductase activity is androgen-dependent, and 17 beta-hydroxysteroid dehydrogenase activity is androgen-resistant. The level and androgen-responsiveness of 5 alpha-reductase in the murine submaxillary gland are not imprinted during the neonatal period by testicular androgens. Circulating androgen may have a cumulative effect on androgen response of 5 alpha-reductase.

An explanation for the proportion of carcinomas and sarcomas seen in chemically induced murine submaxillary gland tumors.

Pellets of 1 mg 9,10-dimethyl-1,3-benzanthracene (DMBA) were implanted into the submaxillary glands of 53 male C57BL/6J mice, and groups of mice were autopsied weekly or biweekly thereafter. Histologic evidence of tumor was noticed first at 12 weeks. From 16 weeks onwards, the submaxillary glands of all mice autopsied contained either carcinoma (three animals) or sarcoma (ten animals); 8/9 attempts to transplant these tumors in C57BL/6J mice were successful. Of the resulting eight tumor lines, two carcinomas and two fibrosarcomas were transplanted for over 30 months. Within the first few transplant generations, all four tumors showed an increase in growth rate and in histologic evidence of anaplasticity. For the particular tumors selected for study, the two carcinomas differed from the two sarcomas by growing more slowly, requiring more cells for tumor takes, and possessing a higher immunogenicity. These results may explain the types of tumors generated in submaxillary gland carcinogenesis. Carcinomas appear first, since they develop from the epithelial cells into which the carcinogen is implanted. Later, when fibroblasts wall off the carcinogen, fibroblasts are at risk for neoplastic conversion. Because of the more aggressive nature of the resultant fibrosarcoma cells, sarcomas may overgrow some early carcinomas.

Synergistic growth stimulation of corneal fibroblasts by components of mesodermal growth factor from murine submaxillary glands.

Recycled mesodermal growth factor (R-MGF) is a pool of proteins of 26,000 molecular weight obtained by recycling gel chromatography of male murine submaxillary gland extracts. R-MGF strikingly accelerates corneal stromal wound healing in vivo, fibroblast growth and migration in cultured corneal buttons and is showing here to stimulate stromal fibroblast growth and division in tissue culture. Chromatographic fractionation of R-MGF has yielded several components, none of which has a greater biological potency than the parent R-MGF. In contrast, two components, MGF-I and -II, when recombined synergistically stimulate fibroblast response in tissue culture and organ culture in excess of those obtained with the parent R-MGF. Three MGF components (I, III, and IV) have been purified and are inactive at 10-15 micrograms/ml in organ culture but potently stimulate fibroblast responses when combined in pairs containing 7.5 micrograms of each component. The striking synergism in organ culture suggests that the stimulation of wound healing by R-MGF in vivo may also reflect synergistic action of more than one R-MGF component. Procedures for isolating gram quantities of R-MGF and for the purification of different R-MGF components by ion exchange chromatography are detailed.

A Comparative Study of the Effects of Cyclocytidine and Fluorocyclocytidine on Murine Submaxillary Salivary Glands

The clinical utility of 1-β-D-arabinofuranosylcytosine (araC), NSC 63878, a chemotherapeutic agent active against a number of acute leukemias and lymphomas, is compromised by the deamination of araC to arabinosyluracil, an inactive metabolite. This observation has led to the study of 2,2'-anhydro-l-β-D-arabinofuranosylcytosine (cyclocytidine, cycloC), NSC 145668, a structurally related, potent anti-tumor agent which is less easily deaminated than is araC. The fluorinated derivative of cycloC, 2,2'-anhydro-l-β-arabinofurano-syl-5-fluorocytosine (fluorocyclocytidine, F-cycloC), NSC 166641, also is an active chemotherapeutic in patients with acute leukemias, and may be most useful in subjects who are resistant to araC. The clinical advantages associated with cycloC are limited, however, by severe salivary gland discomfort and hypersalivation in patients receiving this compound. The present study was undertaken to characterize the cytopathologic alterations in murine submaxillary glands following cycloC therapy and to compare these observations with similar data from studies of mice treated with F-cycloC.