Transduction of TAT-HA-beta-galactosidase fusion protein into salivary gland-derived cells and organ cultures of the developing gland, and into rat submandibular gland in vivo.

We have studied the transduction of TAT-HA-beta-galactosidase fusion protein into two cell lines of rat salivary gland origin, A5 and C6-21, into cells of fetal mouse submandibular glands in organ culture, and into rat submandibular gland after retrograde duct injection, using a histochemical method to demonstrate beta-galactosidase activity. Transduction of the fusion protein into A5 and C6-21 cells was concentration- and time-dependent. Therefore, the intensity of the beta-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr.

Passive immunotherapy of mice bearing Ehrlich ascites tumor expressing human, membrane-bound placental alkaline phosphatase.

The objective of our study was to test if a tumor expressing a transgene coding for a membrane-bound protein is amenable to immunotherapy by antibodies to the same protein. To this end, we have established an Ehrlich ascites tumor (EAT) cell line, EAT-DAP, stably expressing human, membrane-bound placental alkaline phosphatase (PLAP) by infecting EAT cells (EATC) with the retroviral vector DAP and selecting neomycin-resistant cells. EATC and EAT-DAP cells grew at similar rates in vitro, and produced ascites tumor in Swiss-Webster mice with similar efficiency.

Retrovirus-mediated gene transfer into rat salivary gland cells in vitro and in vivo.

A retroviral vector DAP that encodes the human placental alkaline phosphatase (PLAP) and the neomycin-resistant gene was used to transduce the salivary gland-derived cell line A5 in vitro and acinar cells in rat submandibular gland in vivo. Expression of the transduced PLAP gene was established by histochemical staining for heat-resistant AP and by determination of enzyme activity. From the in vitro experiments, we concluded that the salivary gland-derived cell line A5 can be infected by the retroviral vector DAP.

Retrovirus-mediated gene transfer into salivary glands in vivo.

In the present report, we show prolonged expression of beta-galactosidase (beta-Gal) in the acinar cells of the submandibular and sublingual glands of rats following retrograde ductal injection of the retroviral vector BAG. To facilitate integration of viral DNA, cell division in the gland was induced by the administration of the beta-adrenergic agonist isoproterenol prior to the delivery of the vector. The frequency of cells stained for beta-Gal was higher if the virus was injected 4-20 hr after the two injections of isoproterenol given 24 hr apart than after the injection of only one dose of the drug.

Expression of the cysteine proteinase inhibitor cystatin C mRNA in rat eye.

Background: Cystatin C, a naturally occurring inhibitor of cysteine proteinases, belongs to family 2 of the cystatin superfamily. While cystatins in general, and cystatin C specifically, are expressed in various cell types and found in biological fluids, cystatins in ocular structures have not been investigated. In the present study, the expression of cystatin C mRNA in the eye of the rat was studied.

Expressions of the genes for cysteine proteinase inhibitors cystatin C and cystatin S in rat submandibular salivary gland.

Rat cystatin S and rat cystatin C are members of family 2 (cystatin) of the cystatin superfamily. All members of the cystatin family inhibit cysteine proteinases to varying degree. The expression of these two inhibitors, which have a 48% similarity at the nucleotide level, was studied in the submandibular gland using reverse transcriptase-polymerase chain reaction (RT-PCR).

Lack of expression of T-kininogen gene in the hearts of untreated and turpentine-injected rats.

Cystatins, inhibitors of cysteine proteinases, are present in rat heart. However, the controls of genes coding for various cystatins in the heart, and the cellular sites of expression of these genes are not known. With a sensitive reverse transcriptase--polymerase chain reaction T-kininogen mRNA was readily detected in the submandibular glands and livers, but not in the hearts, of control or turpentine-injected rats.

Expression of the cysteine proteinase inhibitor cystatin C gene in rat heart: use of digoxigenin-labeled probes generated by polymerase chain reaction directly for in situ and northern blot hybridizations.

Cystatins represent a widely distributed superfamily of cysteine proteinase inhibitory proteins. We investigated the expression of the cystatin C gene, belonging to the family 2 of cystatins, in the hearts of female rats. Using a highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) we have detected cystatin C mRNA in the ventricule and atrium, as well as in liver and submandibular gland.

Cysteine proteinase inhibitor in cultured human medullary thyroid carcinoma cells.

The TT cell line of human medullary thyroid carcinoma, that retains some of the differentiated functions of thyroid C cells including the synthesis and secretion of calcitonin, was found to contain and release into the culture medium cysteine proteinase inhibitor(s), cystatin(s). The major inhibitor, which is similar to, if not identical with, cystatin C, is constitutively released, or secreted, by TT cells. The rate of secretion of cystatin, quantified by titration of inhibition of papain, was stimulated by dibutyryladenosine 3':5'-cyclic monophosphate, forskolin, the calcium ionophore A 23187, and by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA).

Cystatins in human tear fluid.

The activities of cysteine proteinases which include several lysosomal cathepsins are controlled by naturally occurring inhibitory proteins termed cystatins. Cystatins occur both intracellularly and extracellularly in various tissue fluids including tears. Tears were collected by the Schirmer paper strip method from healthy volunteers who had no history or signs of external ocular disease.

Adrenergic regulation of c-fos expression in cultured BC3H1 muscle cells.

Administration of adrenergic agonists induced c-fos mRNA in the salivary glands of the mouse and in the heart of the mouse, rat, and hamster (Barka et al., 1986, Mol. Cell Biol.

Proto-oncogene fos (c-fos) expression in the heart.

Administration of the beta-adrenergic agonist isoproterenol led to a marked rapid increase in the steady-state level of c-fos mRNA in the heart of mice, rats, and Syrian hamsters. Stimulation of c-fos expression by isoproterenol was inhibited by the beta-adrenergic antagonist propranolol. An increase in Ca2+ influx through voltage-dependent calcium channels is probably not required for the activation of the c-fos gene by isoproterenol since the calcium channel blockers verapamil, nifedipine, and diltiazem had no effect on the induction of c-fos by the drug.

Accumulation of c-fos mRNA in slices of mouse submandibular gland incubated in vitro.

Incubation of slices or isolated lobules of murine submandibular gland at 37 degrees C in physiologic solutions or in tissue culture media, or dissociation of the cells by collagenase-hyaluronidase treatment, increased the steady-state level of c-fos mRNA without any additional stimulus. This activation of c-fos expression required the presence of Na+ and K+ but not extracellular Ca2+. It was augmented by depolarizing concentrations of K+ and by veratridine, and inhibited by high concentrations of amiloride.

Beta-adrenergic stimulation of c-fos gene expression in the mouse submandibular gland.

Isoproterenol (IPR), a beta-adrenergic agonist, induces division of acinar cells in the parotid and submandibular glands of adult rodents and produces hyperplastic and hypertrophic enlargements of these organs. We analyzed the effects of IPR on thymidine incorporation, c-fos mRNA levels, and the immunocytochemical localization of c-fos protein in the submandibular glands of adult and of 5- and 14-day-old mice. In the glands of untreated mice c-fos transcripts were not detectable.

Induction of the synthesis of a specific protein in rat submandibular gland by isoproterenol.

Prolonged treatment of rats with the beta-adrenergic agonist, isoproterenol (IPR), produces hypertrophy and hyperplasia of the parotid and submandibular glands. The drug induces the synthesis of several secretory proteins that are absent or occur at very low concentrations in the gland or saliva of the untreated rat. We have measured the relative concentrations of one of these proteins (termed "large mobile" (LM) protein, Menaker et al.

Monoclonal antibodies against rat saliva and salivary gland antigens.

Hybridomas were produced by the fusion of NS1 myeloma cells with spleen cells of a BALB/c mouse immunized with rat submandibular saliva. Growth of hybridomas was evident in 60/96 wells, and colonies secreting antibodies against saliva components were identified in 20 wells by using a solid phase enzyme-linked immunoassay. Cloning of cells from 12 wells yielded originally 43 hybridoma cell lines secreting anti-saliva antibodies.

Transport of 125I-EGF into milk and effect of sialoadenectomy on milk EGF in mice.

Epidermal growth factor (EGF) (urogastrone) is found in high concentrations in mouse and human milk. The origin of milk EGF is unknown. Milk samples were collected from lactating mice 2-6 h after the intravenous administration of a tracer dose of 125I-labeled EGF.

Culture of A-431 human epidermoid carcinoma cells in serum-free medium: effect of culture conditions on the binding of [125I]-epidermal growth factor.

Serum-free culture conditions that permit the continuous growth of A-431 human epidermoid carcinoma cells were developed. In Dulbecco's modified Eagle's synthetic nutritional medium (DME) supplemented with fetuin, insulin, transferrin, biotin, and oleic acid-fatty acid-free bovine serum albumin complex A-431 cells grew at a rate comparable to that observed in the presence of calf or fetal calf serum. Of the factors tested, oleic acid had the most pronounced stimulatory effect on the growth and [3H]-thymidine incorporation of A-431 cells in serum-free medium.

Hormonal regulation of epidermal growth factor and protease in the submandibular gland of the adult mouse.

The structure of the granular convoluted tubules of the mouse submandibular gland is influenced by androgens, adrenal steroids, and thyroid hormones. We wished to investigate the effects of variations in hormonal status on the quantitative and qualitative distribution of two secretory products of these tubules, epidermal growth factor (EGF) and protease. The effects of the thyroid and adrenal glands on EGF content and protease activity of the submandibular glands of adult female mice were studied by RIAs (EGF), enzyme assays (protease), and immunocytochemical methods.

Hypertrophic and hyperplastic effects of thyroxine on the submandibular gland of the mouse.

The nature of the trophic response of the mouse submandibular gland to thyroxine (T4) was examined. Adult female Swiss-Webster mice were given daily subcutaneous injections of T4 (1 microgram/gm body weight) for two or four days; two injections of tritiated thymidine (3H-TdR) were given 24 and 29 hours after the last injection of hormone, and the mice were killed one hour after the last injection of 3H-TdR. One gland was analyzed chemically for DNA content and for incorporation of 3H-TdR, while the other was used to prepare autoradiograms.

Epidermal growth factor, renin, and peptidase in cultured tumor cells of submandibular gland origin.

Dimethylbenz[a]anthracene was injected into the submandibular glands of male Swiss-Webster mice. From tumors obtained, three cell lines were established. Immunocytochemical stainings revealed epidermal growth factor, renin, and peptidase in a significant portion of cultured tumor cells.

Epidermal growth factor-like material in rat submandibular gland.

By using an antiserum specific for mouse epidermal growth factor (EGF), only the granular convoluted tubule (GCT) cells revealed immunochemical staining in rat submandibular glands. There was no regular sexual difference in the frequency or size of immunoreactive cells. Extracts of gland contained an antigen which showed a complete cross-reactivity with mouse EGF in radioimmunoassays.

Stimulated growth of submandibular gland.

The effects of chronic isoproterenol (IPR) administration on the growth of submandibular gland were studied. Treatment of female rats with IPR, 0.02 mg.

Dissociation of rat parotid gland.

Rat parotid gland was dissociated by sequential collagenase and hyaluronidase digestions, chelation with ethylenediaminetetraacetic acid, and mild shearing force to yield predominantly single cells. The isolated acinar cells retained their morphologic characteristics and their amylase activity. The functional integrity of the isolated cells was assessed by measuring their secretory response to isoproterenol, epinephrine, and carbamylcholine and by their ability to incorporate radioactively labeled leucine and thymidine.