Ultrastructure of the acinar cells in the submandibular gland of the nine-banded armadillo

There are two discrete lobes comprising the armadillo subman-dibular gland. These two lobes can be defined grossly, histochemically and morphologically with the light and electron microscope. The minor lobe stains more intensely with PAS and AB. When viewed in the electron microscope, the secretory granules of the acinar cells within this lobe appear mucous-like. The granules of the demilune cells are slightly different in appearance. The secretory granules of the acinar cells in the major lobe contain many dense foci embedded in a fibrillar matrix, a substructure not described previously. The demilune cells of this lobe contain secretory granules with a mucous-like structure which is consistent throughout the entire lobe. As in the minor lobe, these demilune cells stain very intensely with PAS and AB.     

Isolation and characterization of a protein corresponding to mKlk-11 clone from male mouse submandibular gland

A protein corresponding to the predicted genomic sequence of clone mKlk-11 has been characterized from mouse submandibular gland. This protein was purified by reverse-phase high-performance liquid chromatography from the submandibular glands of normal and hypertensive mice. The protein was not detected in the submandibular gland of mice selected for low blood pressure. It consists of three fragments starting at the residues 1, 98, and 141 of the predicted sequence of clone mKlk-11. The cleavage of-lactoglobulin (between residues 20 and 21, Tyr-Ser, and 40 and 41, Arg-Val) and a synthetic renin substrate tetradecapeptide (residues 4 and 5, Tyr-Ile) by the protein corresponding to clone mKlk-11 showed both tryptic- and chymotryptic-type cleavages. The possibility of this protein's involvement in the regulation of local blood flow is raised.     

Cell-free translation of proline-rich protein mRNAS from human submandibular gland

RNA was isolated from a human submandibular gland and separated into poly A-enriched and poly A-deficient fractions by chromatography on oligo (dT) cellulose. Both of these RNA fractions stimulated methionine incorporation into polypeptides in a reticulocyte lysate translation system. Two in vitro translation products templated by poly A-enriched mRNA were isolated by immunoprecipitation with immune serum directed against human salivary anionic proline-rich protein I. These polypeptides were shown to be precursors of proline-rich proteins on the basis of Mr, affinity for the antiserum, and preferential incorporation of proline. This study is the first to demonstrate cell-free translation of the mRNAs for human proline-rich salivary protein precursors.     

Studies on chromatin-associated protein phosphokinase of submandibular gland from isoproterenol-treated rats

Water-soluble chromatin from rat submandibular gland nuclei was isolated, and had a DNA: RNA:protein ratio of 8:1:20. The spectral properties of this preparation were similar to those described for chromatins from other tissues. The rat submandibular gland chromatin possessed protein phosphokinase activity. It was able to incorporate ³²P from [γ-³²P]-ATP into chromatin proteins, and into dephospho-phosvitin. The chromatin-associated protein phosphokinase activity (measured with dephospho-phosvitin as substrate) required Mg²⁺, Na⁺ or K⁺ and dithiothreitol for optimal activity. A single injection of isoproterenol influenced the activity of this enzyme system, so that it was decreased at 2 h, showed a transient increase at 4 h, and a large increase at 10–16 h after the injection. This event appears to precede the increase in ribosomal RNA induced by Ipr [13]. By 48 h the chromatin-associated protein kinase returned to the normal control levels. These changes appeared to be commensurate with the corresponding alterations in the non-histone acidic protein complement of these chromatins. Actinomycin D or cycloheximide, when administered 30 min prior to isoproterenol, blocked the increase in chromatin-associated protein kinase at 4 as well as 10 h after the injection of isoproterenol. Injection of pilocarpine did not influence the chromatin-associated protein phosphokinase activity. Dichloroisoproterenol appeared to be antagonistic to the influence of isoproterenol in mediating changes in chromatin-associated protein kinase. The results suggest that the isoproterenol-induced increase in chromatin-bound protein phosphokinase which precedes the increase in RNA synthesis is related to the eventual onset of DNA synthesis in rat submandibular gland stimulated by isoproterenol. The chromatin-bound protein phosphokinase activity (or activities) may have a regulatory role on gene action, mediated through the control of phosphorylation of nuclear non-histone acidic proteins [26].     

Preparation and characterization of armadillo submandibular glycoproteins.

The nine-banded armadillo (Dasypus novemcinctus mexicanus Peters) was chosen for this study so that a comparison could be made of the salivary mucus glycoproteins of an ancient mammalian species with those derived from previously studied, more highly evolved species. Two mucus glycoproteins, armadillo submandibular glycoprotein A and armadillo submandibular glycoprotein B, were prepared from the armadillo submandibular gland by a modification of the method of Tettamanti & Pigman (1968) (Arch. Biochem. Biophys. 124, 41-50). The composition of glycoprotein A is the simplest one among the known mucus glycoproteins. Six amino acids constitute 98.5 mol/100mol of the protein of glycoprotein A and 82 mol/100 mol of that of glycoprotein B. These are serine and threonine (which make up 40-50% of the molar amino acid composition), glutamic acid, glycine alanine and valine. Proline is absent from glycoprotein A and comprises only 2.3% of glycoprotein B. For both glycoproteins, the protein content, as determined by the method of Lowry, Rosebrough, Farr & Randall (1951) (J. Biol. Chem 193, 265-275), with bovine serum albumin as standard, was nearly 60% higher than when determined by the sum of the amino acids. The ratios of total mol of amino acid/total mol of carbohydrate are 1:0.63 for glycoprotein A and 1:0.68 for glycoprotein B, N-Acetylneuraminic acid and N-acetylgalactosamine, in a molar ratio of about 0.35:1.00, are the principal carbohydrates present in both glycoproteins. Neutral sugars seem to be absent from glycoprotein A, but galactose and fucose are present in glycoprotein B. The carbohydrate side chains in glycoprotein A are composed of about two-thirds monosaccharide and one-third disaccharide residues, whereas those of glycoprotein B are more complex. For both glycoproteins, essentially all of the N-acetylgalactosamine was attached O-glycosidically to the hydroxyamino acid residues of the protein core. The linkage of N-acetylneuraminic acid glycoprotein A was extremely sensitive to dilute acid and neuraminidase. Glycoprotein B has chemical properties similar to those of glycoprotein A. However, whereas glycoprotein A was susceptible to both Clostridium perfringens and Vibrio cholerae neuraminidases, only the latter enzyme had an effect on glycoprotein B at pH 4.75. Both glycoproteins were homogeneous by cellulose acetate electrophoresis and ultracentrifugal analyses. The apparent mol.wts. of glycoprotein A and glycoprotein B were 7.8 X 10(4) and 3.1 X 10(4) respectively.     

Determination in situ of neutral and acidic fucose-containing oligosaccharides in the bovine submandibular gland

Lectins conjugated to horseradish peroxidase in combination with selected exoglycosidase digestion procedures were used to localize fucoglycoconjugates in the bovine submandibular gland. In particular, sequential treatments were employed to determine the distribution of neutral and acidic fucose-containing oligosaccharides that were previously shown to be present by biochemical techniques. Information was obtained on the distribution of the acidic oligosaccharide A-1a, -Fuc(12)--Gal-(14)--GlcNAc-(13)-[-NeuAc-(26)]-GalNAc-ol, which was sequenced in situ and localized in acinar cells.     

Excision of the submandibular gland by an intraoral approach

To improve the outcome in patients with benign diseases of the submandibular gland, we have developed an entirely intraoral technique for excision of the submandibular gland. This procedure is anatomically safe and can be performed with minimal morbidity. We believe the essential surgical steps are as follows: (1) infiltration with Xylocaine plus epinephrine with an adequate waiting period for hemostasis; (2) careful identification of the submandibular duct/lingual nerve relationship; (3) anterior retraction of the mylohyoid muscle to expose the superficial lobe; (4) superiorly directed, extraoral, manipulation of the submandibular gland; and (5) close and blunt dissection to the gland laterally to avoid injury to the facial artery and vein.     

Structural studies on the carbohydrate units of armadillo submandibular glycoprotein.

The structure of carbohydrate units of the major glycoprotein fraction of armadillo submandibular gland was investigated. Alkaline borohydride reductive cleavage of the glycoprotein resulted in the release of O-glycosidically linked mono- and disaccharide units. The monosaccharide was identified as N-acetylgalactosaminitol, whereas disaccharide contained of N-acetylneuraminic acid and N-acetylgalactosaminitol. Treatment of the native and desialyzed glycoprotein with alpha-N-acetylgalactosaminidase resulted in the removal of 60% and 96% of N-acetylgalactosamine, respectively. No cleavage of this sugar was affected by the action of beta-N-acetylhexosaminidase. Both N-acetylgalactosamine and N-acetylneuraminic acid were susceptible to oxidation with periodate. Analyses of the partially methylated N-acetylgalactosamine derivatives, obtained from the permethylated native glycoprotein, showed the presence of 3,4,6-tri-O-methyl-N-methylacetamidogalactose and 3,4-di-O-methyl-N-methylacetamidogalactose in a ratio of 1 : 0.4. Only 3,4,6-tri-O-methyl-N-methylacetamidogalactose was found in the hydrolysates of permethylated desialyzed glycoprotein. These results together with our previous data on chemical composition of the glycoprotein suggest that about 30% of the oligosaccharide chains consist of NeuAc alpha 2 leads to 6GalNAc alpha 1 leads to O-Thr(Ser) and 70% of GalNAc alpha leads to O-Thr(Ser).     

Basic isozymes of chymotrypsin-like esteroprotease from the mouse submandibular gland

Basic isozymes of chymotrypsin-like esteroprotease from mouse submandibular glands were purified 60-80-fold by a rather simple procedure consisting of CM-Sepharose CL6B chromatography and gel filtration on Sephadex G-100. The purified sample contained three major isozymes (A, B, C) and some minor ones. Their isoelectric points were between pH 10 and 11. The molecular weights of the main isozymes were estimated at 28000 by SDS-polyacrylamide gel electrophoresis. The acidic isozyme (A) separated into two polypeptide chains whose molecular weights were 21500 and 6500. Specific activities of these isozymes using Bz-Tyr-OEt as substrate were comparable to that of bovine pancreatic alpha-chymotrypsin, but they hydrolyzed casein 10 times slower than did alpha-chymotrypsin. The hydrolytic activities of these isozymes on Bz-Tyr-OEt were inhibited by diisopropylfluorophosphate, tosyl-L-phenylalanine chloromethyl ketone and chymostatin, but they were 400 times less sensitive to chymostatin than was alpha-chymotrypsin.     

Purification of an acidic recombinant protein from transgenic tobacco

Tobacco has proven to be a promising alternative for the production of recombinant therapeutic proteins and offers numerous advantages over other plants as a host system. However, the recovery and purification steps needed to obtain a protein at high recovery and purity have not been well investigated. In this study, a process was developed to purify a model acidic protein, recombinant beta-glucuronidase (rGUS) from transgenic tobacco leaf tissue, in three main steps after extraction: polyelectrolyte precipitation, hydrophobic interaction chromatography (HIC), and hydroxyapatite chromatography (HAC). Using this three-step process, up to 40% of the initial rGUS activity could be recovered to near homogeneity as judged by SDS-PAGE. This work demonstrates that acidic recombinant proteins expressed in tobacco may be purified to high yield with high purity in a minimal amount of steps that are suitable for scale-up. Furthermore, the general steps used in this process may suggest that a wide variety of acidic recombinant proteins may be purified in a similar manner from transgenic tobacco or other leafy crops.     

Potassium release from submandibular salivary gland in vitro

Rat submandibular gland slices, incubated in continuously-gassed Krebs-Ringer bicarbonate buffer, were shown to release K⁺ in response to α-adrenergic and muscarinic cholinergic stimulation. The system employed the specific α-, β-adrenergic and cholinergic receptor-blocking agents phentolamine, propranolol and atropine, respectively, in combination with the agonists l-epinephrine and carbamylcholine both of which required the presence of Ca²⁺ for their effect. The introduction of Ca²⁺ into the cell via the ionophore A23187, with all neurotransmitter receptors blocked, resulted in K⁺ release. Ouabain also allowed extensive K⁺ release which was in addition to, and hence independent of, that elicited by epinephrine and carbamylcholine. Ethacrynic acid, a potent inhibitor of salivary secretion in vivo, had no influence on K⁺ movement. K⁺ was released by both physalaemin and an eledoisin-related peptide independently of normal neurotransmitter receptors. The activity of the eledoisin-related peptide did not require the presence of extracellular Ca²⁺. The implication of cyclic GMP at some stage of K⁺ release was suggested by experiments with a phosphodiesterase inhibitor.The results support an hypothesis where the initial stimulus at either α-adrenergic or muscarinic cholinergic receptors causes an immediate permeability change such that Ca²⁺ enters the cells resulting in K⁺ release. The loss of K⁺ is quickly countered by the ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$) ATPase which would be activated by the lowered intracellular K⁺ levels.     

Binding protein for 5 alpha-dihydrotestosterone in mouse submandibular gland.

The changes in the levels of the binding protein for 5 alpha-dihydrotestosterone in cytoplasmic extract of the submandibular glands during development were compared in male and female mice using a DEAE-cellulose filter assay. The binding protein was first detectable 5 days after birth in both sexes, at a time coincident with androgen-independent cytodifferentiation of the convoluted tubular cells in the submadibular gland. The level of the binding protein in female mice was maintained at 5 pmol/mg protein after birth, whereas in males it began to decrease from 3 weeks after birth with inccrease in serum testosterone, becoming much less than a quarter of the level in females or immature mice by 4 weeks after birth. However, after castration, the level of detectable binding protein in mature male mice increased within 7 days to the same level as that in females or immature mice. This suggests that the low binding capacity for exogenous hormone in mature male mice is due to occupancy of the binding sites by endogenous hormone.     

Binding protein for 5α-dihydrotestosterone in mouse submandibular gland

The changes in the levels of the binding protein for 5α-dihydrotestosterone in cytoplasmic extract of the submandibular glands during development were compared in male and female mice using a DEAE-cellulose filter assay. The binding protein was first detectable 5 days after birth in both sexes, at a time coincident with androgen-independent cytodifferentiation of the convoluted tubular cells in the submandibular gland. The level of the binding protein in female mice was maintained at 5 pmol/mg protein after birth, whereas in males it began to decrease from 3 weeks after birth with increase in serum testosterone, becoming much less than a quarter of the level in females or immature mice by 4 weeks after birth. However, after castration, the level of detectable binding protein in mature male mice increased within 7 days to the same level as that in females or immature mice. This suggests that the low binding capacity for exogenous hormone in mature male mice is due to occupancy of the binding sites by endogenous hormone.     

Phenylephrine protects autotransplanted rabbit submandibular gland from apoptosis

Submandibular gland (SMG) autotransplantation is an effective treatment for severe keratoconjunctivitis sicca. Our previous studies have shown that phenylephrine attenuates structural injury and promotes cell proliferation in autotransplanted rabbit SMG. However, the mechanism by which phenylephrine reduces the injury has not been fully evaluated. In this study, we investigate the ability of phenylephrine to inhibit apoptosis in autotransplanted rabbit SMG. We observed that apoptosis occurred in the early phase of SMG transplantation and that phenylephrine treatment protected transplanted SMG from apoptosis. Furthermore, we found that phenylephrine could significantly upregulate the expression of Bcl-2, downregulate the expression of Bax, and inhibit the activation of both caspase-3 and p38 mitogen-activated protein kinase in autotransplanted SMG. Therefore, the cytoprotective effects of phenylephrine on autotransplanted SMG may be a novel clinical strategy for autotransplanted SMG protection during the early postoperative stage of transplantation.