Murray R. Robinovitch

Changes in Rat Parotid Salivary Proteins Induced by Chronic Isoproterenol Administration

Changes in the rat parotid gland and its secretion, brought about by chronic isoproterenol administration, were studied. In addition to the expected enlargement, morphological and biochemical analyses of the glands showed evidence of changes in the secretory components. Chromatographic and electrophoretic experiments revealed both qualitative and quantitative changes in the secretory proteins.

The protein composition of rat parotid saliva and secretory granules.

Rat parotid saliva was collected by surgical cannulation of the ducts and stimulation with pilocarpine; The secreted salivary proteins were resolved on columns of DEAE-Sephadex into five major Fractions, I-V, which were characterized by polyacrylamide disc gel electrophoresis, amino acid analyses and enzymatic assay. Rat parotid secretory granules were isolated by density gradient centrifugation and lysed in hypotonic buffers. Granule content proteins were resolved and examined by the same techniques as for secreted proteins. In both experiments, Fraction I contained RNAase and a major unidentified protein, M1, Fraction II contained the isoenzymes of amylase; DNAase was present in Fraction III and, to a lesser degree, in Fraction IV. The proportions of the enzyme-containing peaks were the same in saliva and granule contents. Fractions IV and V contain proteins of unknown function; Fraction IV contains exceptionally high levels of glutamic acid, glycine and proline in its protein moieties and approx. 6-8% neutral sugars.

Therapeutic effects of daily or weekly chlorhexidine rinsing on oral health of a geriatric population

The effects of a chlorhexidine rinse on salivary Streptococcus mutans, Lactobacillus, and Candida albicans counts and on periodontal conditions (gingival index, plaque index, pocket depths) were studied in 42 elderly subjects. Under supervision, they rinsed either daily or weekly for 6 weeks with a 0.12% chlorhexidine solution (Peridex). Saliva samples were taken for chemical and microbiologic examinations, and periodontal conditions were assessed at baseline, week 6, and 6 weeks after final rinse. Significantly lower S. mutans counts were found at week 6 for both rinsing groups (p less than 0.001). Lactobacillus and Candida counts were also generally lower at week 6, with the clearest improvement among persons with the highest counts of bacteria and yeast. Periodontal conditions were improved at week 6 (p less than 0.001) in both groups. Such improvements were not maintained 6 weeks after the rinsing regimen was completed. At baseline poor oral conditions were noticed, which placed most of the subjects at risk for tooth decay and periodontal disease. Without any other dental procedures but daily or weekly supervised rinsing, oral conditions were improved and this risk was reduced. Daily rinsing was not superior to weekly rinsing with 0.12% chlorhexidine.

Anti-infectivity activity of human salivary secretions toward human immunodeficiency virus.

The purpose of this investigation was to adapt an MT-2 cell syncytium-forming assay for measuring anti-infectivity activity of salivary secretions toward HIV and to determine the distribution of this activity in a population of healthy adult subjects. Whole saliva samples were collected from 27 volunteers, who reported that they did not belong to any group at high risk for HIV infection, and tested for anti-infectivity activity using the syncytium-forming assay. Nine of these subjects were subsequently retested on one or more occasions to assess the variability in appearance of this activity. Parotid and extraparotid salivas of six subjects were also tested. Samples were frozen immediately after collection and submitted in blinded fashion for quantitation of their anti-HIV activity using a syncytia-forming MT-2 cell assay or the p24 antigen ELISA. Nine out of the 27 subjects showed detectable anti-HIV infectivity activity. One parotid sample and one extraparotid sample out of four from subjects with positive whole salivas were positive and none of the parotid or extraparotid samples from two subjects with negative whole salivas were positive. The inhibitory activity ranged from 0.5 to 1 log 10 TCID50/ml and could not be correlated with total protein content in saliva or any specific electrophoretic component. Filtration of the saliva through an Amicon 10 filter before incubation with the virus abolished the activity. Similar studies using two other biological fluids, urine and cerebrospinal fluid, revealed no anti-HIV infectivity activity. These findings confirm the presence in saliva of inhibitory activity directed toward HIV.

Isolation and characterization of the basic proline-rich proteins from rat parotid saliva

Five fractions of basic proline-rich proteins were isolated from rat parotid saliva, obtained by surgical cannulation of the ducts. The purification procedures employed DEAE-Sephadex to isolate a heterogeneous break-through fraction containing the basic proline-rich proteins, followed by gel filtration on Sephadex G-200 to separate the high molecular weight glycoprotein, fraction A, from the other basic proline-rich proteins which were resolved into four additional fractions, SP-1 to SP-4, by ion exchange chromatography on SP-Sephadex. The proteins differed in their amino acid composition and content of neutral and amino sugars. All the proteins were characterized by a high proportion of proline (approx. 40 mol per cent) and glycine (11-23 mol per cent). Four of the fractions were also enriched in glutamic acid/glutamine (19-26 mol per cent). The exception was fraction SP-4, which contained lower levels of glutamic acid/glutamine and has no counterpart in human basic proline-rich proteins. Fraction A, the basic glycoprotein, was heavily glycosylated (59 mol per cent), whereas SP-2 and SP-4 were less glycosylated. Fractions SP-1 and SP-3 contained low levels of neutral and amino sugars. Basic proline-rich proteins constitute a smaller percentage of the total protein in rat parotid saliva than they do in human parotid saliva (10.5 versus 40 per cent). Rat basic glycoprotein fraction constitutes less than 1 per cent whereas the human glycoprotein fraction constitutes 17 per cent. Rat basic proline-rich proteins appear to be larger and less basic than most of the human basic proteins, and they resolve into fewer protein fractions (4 versus 9) with SP-Sephadex chromatography.

Isolation and partial characterization of two populations of secretory granules from rat parotid glands

SummaryA method is described for the isolation of two populations of secretory granules from rat parotid glands utilizing differences in their sedimentation characteristics. The granule preparations were analyzed for homogeneity by electron microscopy and chemical analyses. The soluble contents of both types of granules were obtained by hypotonic lysis, and the proteins compared by SDS-PAGE and ion exchange-gel filtration chromatography. Both populations of secretory granules appear to have the same protein composition as that of the parotid saliva. The secretory granules with the smaller apparent buoyant density became labelled with radioactive leucine earlier than the heavier granules when a pulse of this amino acid was supplied to a gland slice system. The lighter granules appear to represent an earlier stage in maturation.

Demonstration of a class of proteins loosely associated with secretory granule membranes

It is shown, in this study, that rat secretory granule membrane preparations, as prepared by the method of Amsterdam et al. [(1971) J. Cell Biol. 50, 187-200], contain a protein fraction which is removed by washing in isotonic medium. This fraction contains unusually high levels of Pro, Gly and Glx, and appears to label rapidly if the rats are pulsed with [14-c] amino acids prior to removal of the glands. The fraction, which may represent specifically adsorbed secretory protein(s) or peripheral membrane protein, is significant to investigators using this model system to study secretory phenomena.

Preservation of the secretory granules of rat parotid gland for electron microscopy

Abstract Fixation of rat parotid gland for electron microscopy with osmium tetroxide, in several different buffer systems, results in imperfect preservation of the acinar secretory granules. This is obviated by prior fixation in glutaraldehyde. In the latter instance, two types of secretory granules can be discerned: one associated with the Golgi regions, and the other located primarily in the apical cytoplasm. The findings raise the question as to whether osmium tetroxide, without prior fixation with aldehydes, brings about the solubilization of the contents of the secretory granules.

Isolation and partial characterization of secretory granule membranes from the rat parotid gland

Abstract Secretory granule fractions prepared by the Urografin gradient method of Kirshner et al . ( Analyt. Biochem . 52 , 589–594) demonstrate an impressive degree of purity as judged by electron-microscopic appearance and chemical and enzymic analyses. Granule membrane preparations, obtained by hypotonic lysis of the secretory granules followed by simple sedimentation of the particulate material, were seen to be significantly contaminated by mitochondria, unlysed granules and other debris. In an effort to improve the purity of the membranes, lysed granules were subjected to discontinuous sucrose-density centrifugation, resulting in the recovery of 3 fractions, one of which contained membrane fragments, free of contamination by other organelles and debris. This membrane fraction, as well as the other two fractions, were characterized with respect to ultrastructural appearance, amino-acid composition and presence of loosely associated proline-rich proteins. The results indicate that previously reported characteristics of rat parotid secretory granule membranes are inaccurate due to the organelle contamination demonstrated in this study.

Lysosomal enzymes in relation to the isoproterenol-induced secretory cycle in rat parotid gland☆

Abstract Two lysosomal enzymes, cathepsin D and acid phosphatase, were detected in significant amounts in the lysosome-containing subcellular fractions of rat parotid tissue and found to have dissimilar distributions in these fractions. The total levels of these enzymes were measured at various times throughout a complete secretory cycle induced synchronously by fasting rats overnight and administering isoproterenol at time zero. The results showed a 30% increase in cathepsin D activity in the glands by 10 h post-stimulation, and a 20% decrease in acid phosphatase activity 7 h after stimulation. These results suggest that there are cyclic changes in lysosomal enzymes during the secretory cycle of this gland, but that these changes are complex ones and cannot be related to specific cellular processes at this time.