Maija I. Mednieks

Protein expression in salivary glands of rats with streptozotocin diabetes

Diabetes mellitus (DM) is a widespread disease with high morbidity and health care costs. An experimental animal model was employed, using morphological and biochemical methods, to investigate the effects of DM on the expression and compartmentation of salivary gland proteins. The distribution of proline‐rich proteins (PRP), submandibular mucin (Muc10) and the regulatory (RI and RII) subunits of cyclic AMP‐dependent protein kinase type I and type II was determined in the parotid and submandibular (SMG) glands of rats treated with streptozotocin. Quantitative immunocytochemistry of secretory granules in diabetic glands revealed decreases of 30% for PRP in both the parotid and SMG, and a 40% decrease in Muc10 in the SMG. Immunogold labelling showed that RII decreased in nuclei and the cytoplasm in diabetic acinar cells while labelling of secretory granules was similar in control and diabetic parotid. Electrophoresis and Western blotting of tissue extracts of two secretory proteins showed that the response to DM and insulin treatment was gland specific: PRP showed little change in the SMG, but decreased in the parotid in DM and was partially restored after insulin treatment. Photoaffinity labelling showed only RI present in the SMG and mainly RII in the parotid. The results of this and previous studies demonstrating highly specific changes in salivary protein expression indicate that the oral environment is significantly altered by DM, and that oral tissues and their function can be compromised. These findings may provide a basis for future studies to develop tests using saliva for diabetic status or progression in humans.

Growth of sebaceous cells in monolayer culture

SummaryRat preputial cells were grown in an epithelial cell primary monolayer culture system identical to that used for culturing epidermal cells, which were studied for comparison. Despite similar appearance when observed by phase contrast microscopy, other features identified the preputial cells as a unique epithelial cell population. Preputial cells grew as a relatively small number of large colonies, formed domes before confluence, and expressed a specific acinar keratin, K4, which had previously been found in human sebaceous glands. In addition, preputial cells formed fewer cornified envelopes than epidermal cells, too few to discern the reduction of envelope formation by retinoic acid treatment in vitro which was found in epidermal cells. Rat preputial cells in monolayer culture, therefore, are a promising model for studying the effects of hormones on sebaceous cell growth and differentiation.

Site-selective 8-C1-cAMP which causes growth inhibition and differentiation increases DNA (CRE)-binding activity in cancer cells

Control mechanisms of normal differentiation are disrupted in cancer cells but can be restored by treatment with site‐selective cAMP analogs. The cellular events associated with such changes entail compartmental redistribution of the cAMP‐dependent protein kinase type II regulatory subunit, RIIβ. The results of this study indicate that the molecular mechanisms of action involve changes in specific DNA‐binding activity of putative transcription factors. Gel retardation analyses revealed that nuclear extracts from cells of various human cancer cell lines [colon cancer (LS‐174T), gastric cancer (TMK‐1), and leukemia (K‐562)] and rodent pheochromocytoma (PC12) show a concentration‐dependent increase in binding activity to a synthetic DNA that contained the cAMP‐responsive element 5′‐TGACGTCA‐3′ after treatment with 8‐C1‐cAMP. Such an increase in cAMP‐responsive element binding activity was not observed in the 8‐C1‐cAMP‐unresponsive MKN‐1 gastric cancer cells. These findings indicate that the antitumor activity of site‐selective cAMP analogs may reside in the induction of transcription factors that restore normal gene regulation in cancer cells.

The enzymatic synthesis of polyribonucleotides using 5′-adenylylmethylenediphosphonate: A phosphonic acid analog of adenosine triphosphate

Abstract 1. 1. The effects on RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) of 5′-adenylylmethylenediphosphonate, an analog of ATP in which the polyphosphate side chain has been varied, have been studied. 2. 2. Enzymatic synthesis of polyribonucleotides as catalyzed by the enzyme and primed for by DNA and deoxyadenylate-deoxythymidylate copolymer, is shown to occur when 5′-adenylylmethylenediphosphonate is substituted for ATP. The extent of this synthesis is from 30 to 50 % of that observed with the natural substrate, ATP.

Immunogold localization of the type II regulatory subunit of cyclic AMP-dependent protein kinase. Monoclonal antibody characterization and RII distribution in rat parotid cells.

A mouse monoclonal antibody of the IgM class, MAb BB1, specific for the type II regulatory subunit (RII) of cyclic AMP-dependent protein kinase (cAPK), was produced using a purified subcellular protein fraction from rat parotid gland as the original antigen. The antibody immunoprecipitated radioactivity labeled RII from bovine heart cAPK, and from rat and human parotid saliva. Western blot analysis revealed specific binding of the antibody to proteins of 52 and 54 KD in extracts of rat parotid tissue, parotid saliva, and bovine heart cAPK. Immunogold labeling of thin sections of rat parotid gland revealed specific labeling of acinar cell nuclei (especially the heterochromatin), cytoplasm (particularly in areas containing granular endoplasmic reticulum), and the content of secretory granules. Labeling was greatly reduced (approximately 84%) when the antibody was pre-absorbed with an excess of bovine heart cAPK. In duct cells the cytoplasm and nuclei were also labeled, but few gold particles were present over secretory granules. These results provide additional evidence for the presence of nuclear cAPK in rat parotid cells, and confirm previous observations on the presence of cAPK regulatory subunits in acinar secretory granules and saliva. The hybridoma reagent will be used for studies of stimulus responses in the parotid and for immunocytochemical analyses of RII distribution in other secretory tissues.

Amylase and cyclic amp receptor protein expression in human diabetic parotid glands.

BACKGROUND Salivary dysfunction and oral disorders have been described in both type 1 and type 2 diabetes mellitus. However, the cellular and molecular consequences of diabetes on oral tissues remain to be ascertained. The purpose of this investigation was to study, by means of electron microscopy, the morphologic and molecular changes that occur in salivary glands during diabetes. METHODS Biopsy samples of parotid glands were excised from non-diabetic and diabetic (type 1 and type 2) consenting patients and processed by standard methods for routine morphology and electron microscopic immunogold labeling. Specific antibodies were used to determine and quantify the expression of secretory proteins (alphaamylase and the regulatory subunit of type II protein kinase A). RESULTS Morphologic changes in the diabetic samples included increased numbers of secretory granules, and alterations in internal granule structure. Quantitative analysis of immunogold labeling showed that labeling densities were variable among the parotid gland samples. In type 1 diabetes amylase expression was greater than in non-diabetic glands, whereas in type 2 diabetes it was not significantly changed. Expression of type II regulatory subunits was slightly, although not significantly, increased in acinar secretory granules of type 1 diabetic samples and was unchanged in type 2 diabetic samples. CONCLUSIONS Our data show that diabetes elicits specific changes in secretory protein expression in human salivary glands, thus contributing to the altered oral environment and oral disease associated with diabetes.

Immunocytochemical analysis of cyclic AMP receptor proteins in the developing rat parotid gland

UNLABELLED Previous studies showed that regulatory subunits of type II cyclic AMP-dependent protein kinase (RII) are present in adult rat parotid acinar cells, and are secreted into saliva. If the synthesis and intracellular distribution of RII exhibit developmental specificity, then RII can be an indicator of secretory and regulatory activity of salivary glands. OBJECTIVE To determine the expression and distribution of RII in the rat parotid at specific ages representing defined developmental stages. METHODS Parotid glands of fetal, neonatal and adult rats were prepared for morphologic and immunocytochemical study. The cellular distribution of RII was studied using light microscopic immunogold silver staining with anti-RII, and its intracellular distribution using electron microscopic immunogold labeling. RESULTS In utero, parotid RII levels were low; 5-18 days after birth, labeling of secretory granules and cytoplasm rose to a peak, followed by a rapid decrease in both compartments at 25 days. At 60 days, granule labeling increased to levels near those at 18 days, whereas cytoplasmic labeling remained low. Nuclear labeling was highest during the first 3 weeks after birth, and then declined. CONCLUSIONS The higher nuclear and cytoplasmic labeling during the neonatal period may reflect RII involvement in acinar cell differentiation. The accumulation of RII in secretory granules is similar to the pattern of the major salivary proteins, amylase and PSP. The redistribution of RII in these compartments during development may reflect changing gene expression patterns, and may be useful for identification of genetic or metabolic abnormalities.

Reaction of [14C]cyanide with hexosamines, acid polysaccharides and some related compounds

Cyanohydrin formation with [14C]cyanide was studied with N-acetylglucosamine, galactose, mannose, glucose, fucose, glucuronate, hyaluronate hexasaccharide, chondroitinsulfate and hyaluronate. With the simple compounds the reaction approximated second-order kinetics. The monosaccharides and the hyaluronate hexasaccharide took up the theoretical amount of cyanide when incubated with an excess of K14CN. However, the chondroitin sulfates took up more than expected from their number average molecular weights. Similar results were obtained with hyaluronate, suggesting that at high cyanide concentrations some breakdown of the polysaccharides may occur. The method of cyanide labeling and counting of mucopolysaccharides and related substances appears to be suitable for studies of enzymic and non-enzymic degradation of polysaccharides.

Localization of cystic fibrosis transmembrane conductance regulator signaling complexes in human salivary gland striated duct cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent protein kinase (PKA)-regulated Cl(-) channel, crucial for epithelial cell regulation of salt and water transport. Previous studies showed that ezrin, an actin binding and A-kinase anchoring protein (AKAP), facilitates association of PKA with CFTR. We used immunohistochemistry and immunogold transmission electron microscopy to localize CFTR, ezrin, and PKA type II regulatory (RII) and catalytic (C) subunits in striated duct cells of human parotid and submandibular glands. Immunohistochemistry localized the four proteins mainly to the apical membrane and the apical cytoplasm of striated duct cells. In acinar cells, ezrin localized to the luminal membrane, and PKA RII subunits were present in secretory granules, as previously described. Immunogold labeling showed that CFTR and PKA RII and C subunits were localized to the luminal membrane and associated with apical granules and vesicles of striated duct cells. Ezrin was present along the luminal membrane, on microvilli and along the junctional complexes between cells. Double labeling showed specific protein associations with apical granules and vesicles and along the luminal membrane. Ezrin, CFTR, and PKA RII and C subunits are co-localized in striated duct cells, suggesting the presence of signaling complexes that serve to regulate CFTR activity.

Secretory proteins characteristic of environmental changes in cellular signal transduction: Expression in oral fluid

Past studies have shown that both hypo- and hyper-gravity have significant consequences on a variety of tissues and organ systems. It is not known if the effects of environmental stimuli such as altered gravity are beneficial or detrimental, and if the effects can be prevented or reversed. Animal experiments from the Space Lab and Cosmos missions indicate that events that are mediated by cyclic AMP, such as cellular responses to catecholamine and peptide hormone action, are significantly altered in a number of tissues as a consequence of space flight. A secretory cyclic AMP-receptor protein (cARP), is present in saliva, and can serve as an indicator of individual responses to physiologic and environmental stress. Animal experiments have shown that the hypergravity component of space flight is a significant stress factor. In humans, cARP levels in each individual are constant under normal conditions, but elevated after acute stress. Additionally, the levels of cARP in secreted saliva can be compared to tho...