Protein Ingredient in Saliva on Oral Dryness Patients Caused by Calcium Blocker.

Oral dryness as a side effect of certain drugs is increasing. The aim of this study was to examine the change of the protein ingredient in saliva of oral dryness patients caused by calcium blocker. Six patients taking calcium blocker and six healthy elderly were enrolled. Unstimulated salivary flow rate, protein concentration, and flow rate of protein were measured and compared between the patients taking calcium blocker and healthy elderly. iTRAQ (Isobaric Tag for Relative and Absolute Quantitation) proteomic analysis was performed to extract the salivary protein changed in patient taking calcium blocker, and the intensities of Western blotting products were quantified (unpaired t-test). Unstimulated salivary flow rate was significantly lower on patients taking calcium blocker (p < 0.01). Protein concentration tended to be higher and the flow rate of protein tended to be lower on patients. As the result of iTRAQ proteomic analysis, calmodulin-like protein 3, glutathione S-transferase P, and keratin type I cytoskeletal 13 increased characteristically in patient taking calcium blocker, and the expression in calmodulin-like protein 3 was significantly larger (p < 0.01). The results of this study indicated that calmodulin-like protein 3 increased in patients taking calcium blocker and could be a salivary biomarker for oral dryness caused by calcium blocker.

Antigen-presenting cells in parotid glands contain cystatin D originating from acinar cells.

Cystatin D encoded by Cst5 is a salivary classified type II cystatin. We investigated the dynamism of cystatin D by examining the distribution of cystatin D protein and mRNA in rats, to identify novel functions. The simultaneous expression of Cst5 and cystatin D was observed in parotid glands, however in situ hybridization showed that only acinar cells produced cystatin D. Synthesized cystatin D was localized in small vesicles and secreted from the apical side to the saliva, and from the basolateral side to the extracellular region, a second secretory pathway for cystatin D. We also identified antigen-presenting cells in the parotid glands that contained cystatin D without the expression of Cst5, indicating the uptake of cystatin D from the extracellular region. Cystatin D was detected in blood serum and renal tubular cells with megalin, indicating the circulation of cystatin D through the body and uptake by renal tubular cells. Thus, the novel dynamism of cystatin D was shown and a function for cystatin D in the regulation of antigen-presenting cell activity was proposed.

Gene expression profiles of the three major salivary glands in rats.

The protein components of saliva reflect the condition of the whole body as well as the salivary glands. The aim of this study is to characterize the gene expression profiles in each of the rat major salivary glands-the parotid, submandibular, and sublingual glands. Gene expression was analyzed using DNA microarrays, and observed differences in expression of representative genes were confirmed by quantitative, real-time polymerase chain reaction. Among the glands, the contribution to the high expression of genes encoding various proteins, specifically mucin 10, proline-rich glycoproteins, proline-rich protein 2, proline-rich proteoglycans, cystatin 10, amylase, deoxyribonuclease I, and von Ebner's gland protein, was significantly greater in the parotid gland than the other glands. The submandibular and sublingual glands had similar gene expression profiles that differed from profile of the parotid gland. For example, the genes encoding mucin 19 and ovomacroglobulin were highly expressed only in the submandibular and sublingual glands. In summary, we characterized gene expression in the rat major salivary glands and provided basic information on salivary gland marker proteins.

Identification of cysteine proteases and screening of cysteine protease inhibitors in biological samples by a two-dimensional gel system of zymography and reverse zymography.

We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the first-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic acid (TCA) fixation. Protease activity in the 2D-gel was visualized as transparent spots where gelatin substrate was digested after commassie brilliant blue (CBB) staining. Some of the transparent spots from the skin mucus extract of rainbow trout were determined to be a cysteine protease through use of E-64 or CA-074. In the reverse zymography technique, the gel was incubated with papain solution at 37 degrees C for 18 h. Cysteine protease inhibitors from broad bean seeds were detected as clear blue spots after CBB staining. The amino (N-) terminal sequences of four papain inhibitor spots thus detected were demonstrated to be identical to that of favin beta chain, a broad bean lectin. Taken together, our system can be considered to be an efficient technique for discovering and characterizing new proteases and protease inhibitors in biological samples. This is the first report describing a 2D-gel system of zymography and reverse zymography.

Evidence for inclusion of a segment of Escherichia coli genomic DNA in bovine tooth germ mRNA encoding salivary proline-rich protein P-B.

In the course of cloning of bovine cDNA for proline-rich protein (PRP) P-B from bovine tooth germ cDNA, we found that one clone with 662 bp contained a 5'-terminal 393 bp (1-393 bp) sequence essentially identical to that of human P-B cDNA (154-546 in D29833) and bovine P-B cDNA (1-356 bp in AB192573) and a sequence of 233 bp (394-626 bp) highly homologous to the segment of E. coli K12 genomic DNA (365511-365744 in NC000913). Although the latter sequence is contained in the vector pT7Blue, which we used, our results show that this chimeric structure in bovine tooth germ P-B cDNA is not an artifact formed during the cloning process, but intrinsic to the bovine genome since the chimeric structure was detected in bovine tooth germ and bovine genomic DNA.

Influence of aging on the carcinogen-induced protein kinase activity in rat liver cell nuclei.

A carcinogen-induced increase in a protein kinase activity was found in cell nuclei of rat liver. The enzyme was extracted from isolated nuclei with a hypotonic buffer, retained to an anion-exchange column, eluted with 0.15 M NaCl containing solution and to be measured for the activity with casein as the substrate, showing a nature of a casein kinase. The change in the activity during the course of aging was studied with 5-, 10-, and 50-week old Wistar male rats. The activity was highest in 5-week-old rat but decreased in 10- and 50-week-old animal. A hepatocarcinogen, thioacetamide, induced an increase in activity in 10-week old rats but rather decreased in 5- and 50-week-old rats. Aging suppresses the activity of this unique enzyme. Thioacetamide abolishes this suppression resulting in an increase in the activity of the enzyme at a certain stage of aging.