Detection of a calmodulin-sensitive cyclic nucleotide phosphodiesterase in rat parotid gland

Calmodulin coupled to Sepharose has provided a rapid and sensitive means of isolating a cyclic nucleotide phosphodiesterase activity which is stimulated by the calmodulin-Ca²⁺ complex, from rat parotid gland. Initial experiments established that phosphodiesterase activity sensitive to calmodulin and Ca²⁺ could not be demonstrated in crude extracts of rat parotid gland or after partial purification of rat parotid phosphodiesterase over DEAE-cellulose. However, it was possible to readily demonstrate the presence of a cyclic nucleotide phosphodiesterase activity regulated by calmodulin if the extracts were first purified by batch ion-exchange chromatography over DEAE-cellulose followed by affinity chromatography with calmodulin coupled to Sepharose. The batch ion-exchange chromatography step removed the major portion of free parotid calmodulin which could compete with calmodulin-coupled Sepharose for the proteins regulated by calmodulin. Thus, by employing an initial chromatography step over DEAE-cellulose to separate phosphodiesterase activity from calmodulin, it was possible to increase the recovery of calmodulin-sensitive phosphodiesterase after affinity chromatography with calmodulin coupled to Sepharose. This approach should be useful for demonstrating the presence of and for purifying other parotid proteins regulated by calmodulin.     

Aggregation of 27 oral bacteria by human whole saliva

Twenty-seven oral strains of the genera Actinomyces (5), Bacteroides (3), and Streptococcus (19) were tested for aggregation by human whole saliva, as well as the effect of culture medium, Ca-ions, and bacteria concentration thereupon. Of the media tested, GF-broth gave rise to less interference by autoaggregation or higher aggregation titers than BHI and TSB, and was used throughout this study. In most cases, Ca-ions (1 mM) only enhanced the rate of induced aggregation, whereas raising the bacteria concentration increased the rate of both induce- and autoaggregation. The final titers, ranging from 1–64, were hardly affected by these parameters, except those of S. rattus HG 59 and S. mutans HG 199, which were respectively increased and decreased by Ca-ions. Saliva-induced aggregation was observed for 21 strains of A. viscosus, A. naeslundii, A. israelii, B. gingivalis, B. intermedius, S. cricetus, S. mutans, S. rattus, S. sanguis, and S. sobrinus, mostly within 15 min to 3 h. Seventeen of these strains also showed autoaggregation, usually well after the onset of induced aggregation. Any potential induced aggregation of B. gingivalis HG 91 was always masked by autoaggregation, as well as that of the S. mutans strains under a particular set of conditions. The aggregation rate and titer varied considerably in a mutually unrelated and strain-dependent way. These microtiterplate data were matched by the 5 spectrophotometric patterns observed for saliva-bacterial interaction, which moreover, gave the better differentiation between induced and autoaggregation. In conclusion, most strains tested can show rapid saliva-induced aggregation in a strain-dependent way, yet strongly affected by the experimental conditions and interference from autoaggregation.     

Coupled Na+/H+ exchange in rat parotid basolateral membrane vesicles

Summary pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H⁺ gradient (pH_in=6.0, pH_out=8.0)²²Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pH_in=pH_out=6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K ₁ =1.6 m) while the transport inhibitors furosemide (1mm), bumetanide (1mm) SITS (0.5mm) and DIDS (0.1mm) were without effect. This transport activity copurified with the basolateral membrane marker K⁺-stimulatedp-nitrophenyl phosphatase. In addition²²Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H⁺ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration cosistent with the existence of a single transport system withK _M =8.0mm at 23°C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na⁺/H⁺ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.     

Identification of G-proteins in rat parotid gland plasma membranes and granule membranes: presence of distinct components in granule membranes

We have identified by immunoblotting and ADP-ribosylation by cholera toxin and pertussis toxin the presence of Mr 43 and 46 KDa G_s, and 39 and 41 KDa G_i;.. subunits in rat parotid gland plasma membranes but not in granule membranes. A Mr 28 KDa polypeptide that served as substrate for ADP-ribosylation by both cholera toxin and pertussis toxin was present exclusively in granule membranes. Photoaffinity crosslinking of [-³²P]GTP showed the presence of high molecular weight GTP-binding proteins (Mr 160,100 KDa) in granule membranes. Six low molecular weight GTP-binding proteins (Mr 21–28 KDa) were differentially distributed in both plasma membranes and granule membranes. The present study identifies various GTP-binding proteins in rat parotid gland plasma membranes and granule membranes, and demonstrates the presence of distinct molecular weight GTP-binding proteins in granule membranes. These granule-associated GTP-binding proteins may be involved in secretory processes.     

Calcium entry in rat parotid acinar cells

Conclusions While it is generally accepted that Ca²⁺ plays an important regulatory role in the physiology of a number of non-excitable cells, the mechanisms which regulate intracellular [Ca²⁺ are far from well established. Ca²⁺ transporting mechanisms which distribute Ca²⁺ intracellularly as well as those which allow influx of extracellular Ca²⁺ are involved in mediating intracellular Ca²⁺ homestasis. In this paper we have described recent studies on the regulation of the Ca²⁺ influx system in the data, it appears that the process of Ca²⁺ entry is extremely complex and may involve several levels of regulation. Understanding the molecular basis of these regulatory mechanisms presents a challeging problem for future studies.     

Ion accumulation in the cell walls of rice plants growing under saline conditions: evidence for the Oertli hypothesis

Abstract. When plants of rice ( Oryza saliva L.) are subjected to mildly saline (50mol m⁻³ NaCl) conditions, the leaves show symptoms of water deficit, even though ion accumulation has been more than sufficient to adjust to the decrease in external water potential. After a few days of exposure to salt, there is a negative correlation, in a population of leaves, between the leaf water concentration (g water per g dry weight) and their sodium concentration (mmol Na per g dry weight). Ion concentrations in the cell walls and the cytoplasm of cells of plants grown in low salinity were measured by X-ray microanalysis. The NaCl concentration in solution in the apoplast was calculated to be around 600mol m⁻³ in leaves of plants whose roots were exposed to only 50 mol m⁻³ NaCl. This constitutes strong evidence that an important factor in salt damage in rice is dehydration due to the extracellular accumulation of salt as suggested in the Oertli hypothesis. The implication, that changes in tissue ion concentration and solute potentials equivalent to the external medium is not evidence of plant osmotic adjustment to salinity, is discussed.     

Comparative studies of diurnal variations of nitrate reductase activity in wheat, oat and barley

Diurnal variations of in vitro and in vivo (intact tissue assay) nitrate reductase (EC 1.6.6.1) activity and stability were examined in leaves of wheat ( Triticum aestivum L. cv. Runar), oat ( Avcna saliva L. cv. Mustang) and barley ( Hordeum vulgure L. cv. Agneta and cv. Gunillu). Nitrate reductase activity was generally higher for wheat than for oat and barley. However, the diurnal variations of nitrate reductase activity and stability were principally the same for all species, e.g. the high activity during the photoperiod was associated with low stability. All species showed a rapid (30-60 min) increase in the in vitro and in vivo activity when the light was switched on. When light was switched off the in vitro activity decreased rapidly whereas decrease in in vivo activity was slower. These experiments support the hypothesis that an activation/ deactivation mechanism is involved in the regulation of diurnal variations in nitrate reductase activity. Red light enhanced nitrate reductase activity in etiolated wheat and barley leaves. In green leaves, however, the daily increase in nitrate reductase activity was not induced by a brief red light treatment. Indications of different regulation mechanisms for the diurnal variations of nitrate reductase activity among the cereals were not found.     

Neurotransmitter-caused increase in [3H]inositol incorporation into phosphatidylinositol de novo synthesis vs exchange

[3H]inositol and 32Pi were simultaneously incorporated into rat parotid phosphatidylinositol. The ratio of [3H]/32Pi incorporation dropped dramatically following stimulation with muscarinic or alpha-adrenergic agonists and returned to control values following the addition of appropriate antagonists. The drop in [3H]/32Pi ratio can be explained by a rapid increase in de- novo synthesis of phosphatidylinositol following its receptor-mediated breakdown. The change in this ratio also provided evidence for the existence of CDP-DG + inositol in equilibrium phosphatidylinositol exchange reaction in the intact tissue.     

Interaction of the salivary low-molecular-weight mucin (MG2) with Actinobacillus actinomycetemcomitans

Periodontitis is associated with the presence of certain Gram-negative bacteria in the oral cavity, among these Actinobacillus actinomycetemcomitans. In order to determine which types of salivary components interact with A. actinomycetemcomitans two strains (HG 1175 and FDC Y4) were incubated with whole saliva and individual glandular secretions, viz. parotid, submandibular, and sublingual saliva. Immunochemical analysis by immunoblotting of bacteria-bound salivary proteins showed that IgA, the low-molecular mucin MG2, parotid agglutinin, and a 300 kDa sublingual and submandibular glycoprotein, were bound to the bacterial strains tested. In addition, adherence of A. actinomycetemcomitans to salivary proteins in a solid-phase was studied. After electrophoresis and transfer of salivary proteins to nitrocellulose membranes A. actinomycetemcomitans adhered only to MG2. In this assay periodate treatment, mild acid hydrolysis or neuraminidase digestion of the saliva glycoproteins abolished binding of two clinical isolates (HG 1175 and NY 664), suggesting that sialic acid residues on MG2 are involved in the binding. In contrast, adherence of the smooth laboratory strain Y4 was not affected by removal of sialic acid residues or even periodate treatment of MG2.Abbreviations S-IgA Secretory IgA - MG1 high-molecular-weight mucin - MG2 low-molecular-weight mucin - EP-GP extra parotid-glycoprotein - PRPs proline-rich proteins - SNA Sambucus nigra agglutinin - MAA Maackia amurensis agglutinin - PNA peanut agglutinin - UEA Ulex europaeus agglutinin     

儿童唾液中细菌总数的测定

本文将昆明地区１１－１５岁健康儿童９０名按不同的口腔条件分为三组,每组３０人。采用培养基表面主培养主数法,分别测定唾液中厌氧菌和需氧菌细菌总数。结果表明：儿童唾液中细菌总数,牙颌畸形组、正畸组、正常组,三组间结果在统计学无显著差异。