Hendrika van der Noen

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. The tear components were extracted from the filter papers, and used to determine the apparent free cystatin activity and cystatin levels of tears, and for immunoblots. Tears were also collected using capillary tubes for the measurements of cystatins. By titrating papain, a cysteine proteinase, of known specific activity with tear fluid, relatively high levels of apparent free cystatin activity were demonstrated in tears: 28.8 +/- 3.47 (S.E.M.) pmols papain inhibited per mg tear protein (n = 9). The concentrations of cystatins in tear samples were measured by an indirect enzyme-linked immunosorbent assay (ELISA) using antibodies against human salivary cystatin S and purified cystatin S as standard. The ELISAS revealed that tears contain high levels of cystatin-like immunoreactive material, amounting to about 10% of tear proteins. In microgram cystatin S/mg protein the values were: right eye: 94.7 +/- 9.9; left eye: 115.5 +/- 14.8; n = 12. Cystatin levels of tears collected using capillary tubes were comparable: 120.7 +/- 19 micrograms/mg protein (n = 10). Immunoblots of tear fluids revealed a protein of about 14,000 molecular weight which reacted with antihuman cystatin SN monoclonal antibodies. Protein(s) of similar molecular weight were visualized using antibodies against human cystatins S and C. Less abundant additional cystatin-like immuno-reactive proteins were detected by using the two latter antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of secretion of epidermal growth factor and amylase by cyclocytidine

SummaryRadioimmunoassays and immunocytochemical techniques were used to assess the effect of cyclocytidine, an antitumor agent, on the level and localization of Epidermal Growth Factor (EGF) in the submandibular gland of the male mouse. A single intraperitoneal injection of 150 mg/kg of cyclocytidine caused, within 6 h, a degranulation of the granular convoluted tubule (GCT) cells and reduced the concentration of immunoreactive EGF in gland extracts by more than 90 %. This effect was largely abolished by the administration of dibenzyline but not by propranolol, indicating that the secretory effect of the drug on the GCT cells is mediated by α-adrenergic receptors. By immunocytochemical staining EGF was localized to the GCT cells. Immunocytochemical staining revealed the same trends in changes in EGF concentration as the radioimmunoassays. However, even at the peak of the cyclocytidine effect there were cells which retained their secretory granules and apparently their EGF complement. In addition, there was a lobular variation in the secretory response. Cyclocytidine caused a transient increase in the blood level of EGF. Furthermore, it stimulated amylase secretion from the gland, which also involved α-adrenergic receptors. Cyclocytidine will be useful in future analyses of the release of various biologically active substances from the GCT cells of the mouse submandibular gland.

Transduction of TAT-HA-β-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-β-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 β-galactosidase activity. Transduction of the fusion protein into A5 and C6–21 cells was concentration- and time-dependent. Therefore, the intensity of the β-galactosidase staining, which was cytoplasmic, was less after 1 hr of exposure compared to exposures up to 24 hr. However, the fusion protein was transduced into 100% of both types of cultured cells. When explants of mouse fetuses at 13 days of gestation were exposed to the fusion proteins, both epithelial and mesenchymal cells were stained for the enzyme, with a conspicuous accumulation of the reaction product at perinuclear cytoplasmic regions. The histochemical staining of the mesenchymal cells was more intense compared to that seen in epithelial cells. TAT-HA-β-galactosidase fusion protein was also delivered to rat submandibular glands by retrograde duct injection. Histochemical staining for β-galactosidase activity of cryostat sections prepared from the injected glands revealed that the transduction of the fusion protein was also time- and dose-dependent. In the glands of rats sacrificed from 10 min to 1 hr after the retrograde injection, essentially all acinar and duct cells showed cytoplasmic staining. The intensity of the staining then declined, and was not seen in the glands of rats killed 24 hr after the injection of the fusion proteins. These results indicate that a full-length, active TAT fusion protein can be targeted to salivary gland cells both in vitro and in vivo to analyze physiological, developmental, and pathophysiological processes.

STIMULATION OF DNA SYNTHESIS BY ISOPROTERENOL IN RAT SUBMANDIBULAR GLAND DURING POSTNATAL GROWTH

The post‐natal growth of rat submandibular gland and the effect of isoproterenol on this process were studied. Between 2 and 42 days of age the DNA content of the gland increased linearly but the increase in RNA and protein content was more rapid after 29 days of age. The RNA: DNA and protein: DNA ratio increased linearly with age. The proliferative activity, measured by the incorporation of tritiated thymidine, was maximum in the gland of 7‐day‐old rats. It declined steadily to a low level in 42‐day‐old rats. A single injection of isoproterenol had no effect on thymidine incorporation in 2‐day‐old rats. The drug, however, stimulated DNA synthesis in older animals and the degree of stimulation was inversely correlated with the proliferative activity in control rats. Small doses of isoproterenol given to rats for 4 days between 2 and 5 days of age produced a hypertrophy of the submandibular gland. The same treatment between 7 and 10 days of age caused both hypertrophy and hyperplasia of the gland. It is concluded that both the regulation of growth and the regulation of induced cell proliferation are a function of cellular differentiation and that cell proliferation can be induced only in cells that reached a certain degree of differentiation.

Adenylate cyclase activity in rat submandibular gland during postnatal development.

Abstract Adenylate cyclase activity was measured in homogenates of submandibular glands of 1 to 42 day old rats. During this period of time the gland reached its final stage of differentiation. Adenylate cyclase activity was higher in the glands of one day old rats than in those of 7 and 14 day old animals. Between 14 and 28 days of age the enzyme activity more than doubled and approached the level that characterized the glands of adult animals. Fluoride (10mM) stimulated the enzyme activity in all age groups but the stimulation was less in the case of one day old rats as compared to older animals. Isoproterenol (10 −4 M) stimulated adenylate cyclase by 50–60% in the gland of adult rats but had no effect on the enzyme activity in 7 to 28 day old animals. Administration of isoproterenol for 5 days to 9 day old rats increased the weight of the submandibular gland by 70 per cent. Total adenylate cyclase activity increased parallel with the weight of the gland but the specific activity of the enzyme remained unchanged. It is concluded that during the postnatal development of the submandibular gland the rapid increase in adenylate cyclase activity occurs after weaning and it coincides with an accelerated rate of functional differentiation of the acinar cells.

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. In the transduced cells the viral long terminal repeat (LTR) promoter was effective, and the cells expressed heat-stable PLAP which was localized mostly in the plasma membrane and could be released by treatment with bromelain or phosphatidyinositol-specific phospholipase C. A5-DAP cells secreted PLAP into the medium. Clones of A5-DAP cells expressed various levels of the enzyme. The level of enzyme activity in different clones was unrelated to growth rate. Retrograde ductal injection of the viral vector into the duct of the submandibular gland of rats resulted in integration and long-term expression of PLAP gene in acinar cells. Expression of PLAP was seen up to 25 days, the limit of the observation period. To facilitate integration of the viral DNA, cell division of acinar cells was induced by administration of the β-adrenergic agonist isoproterenol before administration of the virus. PLAP was secreted into submandibular saliva. The data support the notion that salivary glands are suitable targets for gene transfer in vivo by a retroviral vector. (J Histochem Cytochem 45:1533–1545, 1997)

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). Northern blot hybridization and in situ hybridization with digoxigenin-labelled DNA probes. Both inhibitors were expressed in the serous acinar cells of the submandibular gland. In accord with previous findings, cystatin S mRNA was induced by the beta-adrenergic agonist isoproterenol. The level of cystatin S mRNA, which was very low in the glands of untreated rats and was demonstrable by RT-PCR but not by Northern blot hybridization, was not altered by acute inflammation produced by turpentine. Neither the administration of isoproterenol nor acute inflammation had any effect on the level of cystatin C mRNA, indicating beta-adrenoreceptors are not involved in the regulation of the cystatin C gene(s) in the submandibular gland. The data indicate that these two closely related genes, expressed in the same cells, are differently regulated. The consequence of this difference in gene regulation on the physiological and pathological roles of these inhibitors remains to be established.

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. Immunocytochemical observations employing a monoclonal antibody to bradykinin, which reacts with kininogens in general, revealed no specific staining in cardiac structures, but a weak staining was apparent in blood vessels and on the surface of endothelial cells of both control and turpentine-injected rats. The monoclonal antibody revealed the presence of kininogens in the acinar cells of the submandibular gland, and, in acute inflammation, in the hepatocytes. These findings suggest that the T-kininogen gene is not expressed in the heart, and the T-kininogen demonstrable in heart extracts derives from the blood. Circulating kininogens are likely bound to endothelial cells, and may be a local source of kinins. In addition, kininogens, as potent inhibitors of cysteine proteinases, may play a role in pathologic conditions of the heart by controlling the deleterious effects of cathepsins released from lysosomes or secreted by macrophages.