Viscoelastic properties of solutions of ovine submaxillary mucin.

The linear viscoelastic and rheological properties of high molecular weight ovine submaxillary mucin (OSM) solution have been investigated in terms of the Newtonian steady-flow viscosity [eta(gamma)], the complex oscillatory viscosity [eta*(omega)], and the storage and loss shear moduli [G'(omega) and G"(omega)]. It was observed that tau(gamma), eta*(omega), and G'(omega) are always higher when OSM is dissolved in 0.1M NaCl than when at the same concentration in 6M GdnHCl. This is consistent with previous observations that submaxillary mucins self-associate in 0.1M NaCl to form large aggregates, which are disrupted in 6M GdnHCl. As the OSM concentration increases, the appearance of a plateau shear modulus indicates the formation of a gel network in both solvents. The results suggest gelation involves specific intermolecular interactions, perhaps due to hydrophobic forces between interdigitated oligosaccharide side chains. The viscoelastic behavior of OSM solution at high concentration is thus similar to that reported in the literature for porcine gastric mucin (PGM). However, the OSM gels are mechanically weaker, having moduli that are an order of magnitude lower than those for PGM gels of comparable concentration. The oligosaccharide side chains of OSM consist of only 1-2 sugar units compared to 10-15 for PGM, but it appears that this is sufficient to allow for intermolecular interaction and the formation of weak gels.

Localization of chromogranins, non-neuron-specific enolase, and different forms of somatostatins in the submandibular salivary glands of mice.

The localizations of chromogranins A, B, and C, neuron-specific enolase (NSE, gamma gamma-type) and non-NSE (alpha alpha-type), and different forms of somatostatins were immunocytochemically identified. The localizations were compared with those of epidermal growth factor (EGF) and nerve growth factor (NGF) in the submandibular salivary glands (SMG) of male mice at five to six weeks of age, with use of a variety of antibodies and the peroxidase-antiperoxidase (PAP) and avidin-biotin complex (ABC) detection methods. In the SMG of male mice, the major chromogranin present was chromogranin A, whereas chromogranins B and C were not detected at these ages by either method. Chromogranin A-like immunoreactivity was located in the granular convoluted tubule (GCT) cells of the SMG, whereas non-NSE immunoreactivity was observed throughout the duct system and in some acinar-associated cells. NSE was not detected in any part of the SMG. The distribution of chromogranin A and somatostatins in the GCT cells was similar to that of EGF and NGF. Our results strongly suggest that chromogranin A and somatostatins, but not chromogranin B or C, may be useful as a means of differentiation of the cells in the duct system of the SMG responsible for the production of biologically-active factors.

Essential fatty acid deficient rats in the study of cystic fibrosis: an X-ray microanalytical and ultrastructural study in chronically reserpinized rats.

Essential fatty acid (EFA) deficiency has been proposed as a major pathogenic mechanism for cystic fibrosis (CF) and EFA-deficient animals have been proposed as an animal model for CF. In the present study, the elemental composition and ultrastructure of the acinar cells of the submandibular and parotid gland of the pancreas of EFA-deficient rats were investigated by X-ray microanalysis and electron microscopy. The effects of EFA-deficiency were compared to changes in these exocrine glands in chronically reserpine-treated rats, an established animal model for CF. EFA-deficiency did not cause any significant changes in the elemental composition of the acinar cells of the submandibular or parotid gland, or of the pancreas. The changes in elemental composition induced by reserpine treatment were only slightly modified by EFA-deficiency, mainly towards normalization. EFA-deficiency resulted in the presence of abnormal, electron translucent, zymogen granules in the parotid gland and in a reduction of the number of zymogen granules in pancreatic acinar cells. Since EFA-deficiency in rats only causes minor changes in structure and elemental composition of salivary glands and pancreas, and does not potentiate the effect of chronic reserpine treatment on these tissues, it is concluded that EFA-deficiency is likely to be of minor importance in the exocrine gland disturbances in CF.

Stimulation of corneal endothelial wound healing in cats by mesodermal growth factor.

Several growth factors have been evaluated for their effects on corneal wound healing but few studies have yet demonstrated an acceleration of endothelial repair in vivo. Mesodermal Growth Factor (MGF) was tested in vivo by making standardized freeze wounds in cat corneas and immediately injecting one of four concentrations of MGF in sterile phosphate buffered saline (PBS), or PBS alone, into the anterior chamber. Seven days later, the animals were sacrificed and the corneas excised and stained. Descemet's membrane and endothelium were dissected and mounted onto glass slides. The wound areas were photographed, measured and compared statistically. Those cats receiving the three lowest doses of MGF had significantly smaller wounds than controls (p less than 0.05).

A simple and rapid purification of kallikrein from rat submandibular gland.

Rat submandibular kallikrein was isolated in an 87% yield by a very quick and simple procedure involving hydrophobic interaction chromatography. Furthermore, that purification method was superior to both aprotinin-affinity chromatography and immunoaffinity chromatography for the purification of rat submandibular kallikrein. The kallikrein purified by hydrophobic interaction chromatography consisted of a number of isoenzymes. The major component of Mr 38,000 seen on SDS-gel electrophoresis was found to be the glycosylated kallikrein, whereas the minor component of Mr 26,000 represented the non-glycosylated enzyme.

Studies of naturally occurring modifications of sialic acids by fast-atom bombardment-mass spectrometry. Analysis of positional isomers by periodate cleavage.

A variety of modifications of sialic acids have been described in nature. There are currently many difficulties in the detection and quantitation of these modified sialic acids from biological sources. We report here that fast-atom bombardment-mass-spectrometry (FAB-MS) of native sialic acids provides specific detection and quantitation of many previously known compounds. Derivatization of the sialic acids by reduction and peracylation under acidic conditions prior to FAB-MS provides further confirmation of their identity and improves the sensitivity of detection. Samples containing as little as 100 ng of a derivatized sialic acid loaded onto the FAB target allowed accurate identification. Mixtures of sialic acids could be analyzed, and minor components were seen, at levels undetectable by other currently known techniques. Analysis of known mixtures of different sialic acids gave reproducible relative signal intensities, indicating that quantitative data can be derived from the FAB-MS spectra. After reduction and peracylation, each sialic acid gave two major molecular ions, corresponding to the fully derivatized linear species and a lactone form, and a minor ion, corresponding to an anhydro form. Lactone formation was minimal in the case of four substituted sialic acids, indicating that the hydroxyl group at the 4-position is involved in lactonization. Differentiation between different positional isomers of the modified sialic acids could be achieved using controlled degradation with periodate, tagging of the fragments with p-aminobenzoic acid ethyl ester under acid reducing conditions, peracylation, and FAB-MS of the derivatized products. We used this FAB-MS strategy to identify a novel sialic acid, 8-O-methyl-7,9-di-O-acetyl-N-glycolyl-neuraminic acid from the starfish Pisaster brevispinus, and to demonstrate the presence of a previously undetected sialic acid, 4,8-anhydro-N-acetyl-neuraminic acid in acid hydrolysates of horse serum. We also use FAB-MS to show that the alkaline conditions traditionally used for analytical de-O-acetylation of sialic acids causes substantial conversion of 4-O-acetylated sialic acids into the same anhydro compound.

A rapid radioimmunoassay for inducible rat submandibular gland cysteine proteinase inhibitor (cystatin).

A highly sensitive and specific radioimmunoassay for the determination of rat family 2 cystatins has been developed. Antibodies against cystatin were elicited in rabbits after coupling the protein to thyroglobulin. Antibodies were specific for rat family 2 cystatin and did not cross-react with human salivary cystatin, rat family 3 cystatin (kininogen) or other salivary proteins. Fifty percent precipitation of 125I-cystatin was obtained with a 1:100,000 dilution of antisera. Optimal conditions for each step of the assay were established from a series of initial experiments. Precipitation of the antigen-antibody complex was achieved with 10% polyethylene glycol in 0.05 M phosphate buffer, pH 7.5 in the presence of 0.1% gamma-globulin. A standard curve constructed by plotting the fraction of bound 125I-cystatin against submandibular gland cystatin in a logit-log mode yielded a straight line for cystatin concentrations between 0.2 and 200 ng. The lowest limit of detection of family 2 cystatin by RIA was 0.2 ng, and is 1000 fold more sensitive than enzyme-inhibition assays and Laurell rocket electrophoresis. Only trace amounts of cystatin were detected in submandibular, sublingual and parotid glands from normal rats. After isoproterenol-stimulation cystatin levels increased dramatically in salivary glands and moderately in kidney and pancreas.

Characterization of monoclonal antibodies to human salivary (glyco) proteins. Cellular localization of mucin, cystatin-like 14 kD protein and 20 kD glycoprotein in the human submandibular gland.

Using the hybridoma technique, monoclonal antibodies (Mabs) have been produced against three different types of human salivary proteins: high molecular weight mucin, a 20 kD glycoprotein and a 14 kD protein, identified as a member of the cystatin family. The Mabs appeared to be highly specific to their antigen in Elisa and immunoblotting tests. The Mabs were of the IgG-1 (against 20 kD glycoprotein) and IgM (against 14 kD protein and mucin) type. For the 14 kD protein and the 20 kD glycoprotein it was demonstrated that they are present mainly in submandibular-sublingual saliva. None of the antigens studied could be localized distinctly in the human parotid gland. In the submandibular gland, the three proteins have a different pattern of localization. The mucins have been detected particularly in the apical part of the mucous acinar cells, the 20 kD glycoprotein mainly in the serous acinar cells and the 14 kD protein in both serous acinar cells and striated duct cells.

Light scattering studies of the effect of Ca2+ on the structure of porcine submaxillary mucin.

The effects of calcium ions on the solution properties of porcine submaxillary mucin (PSM) have been investigated by static and dynamic light scattering. The weight average molecular weights of PSM fractions are unaffected by the addition of up to 0.5M CaCl2: these data are within experimental error of those for solutions in 0.1M NaCl. The distribution of relaxation frequencies derived from the dynamic data shows the existence of two distinct relaxation modes. The average relaxation times have been interpreted to yield the z-average translational diffusion coefficient and the longest intramolecular relaxation time tau1. A plot of tau1 vs the mean value of 1/Rh-3z is linear, and consistent with plots of such data recorded for PSM in 0.1m NaCl and 6M GdnHCl solutions. However, the tau values and the associated results for the mean value of R-1h-1z in 0.5M CaCl2 are smaller than those determined in 0.1M NaCl. This suggests that the conformation of PSM in CaCl2 solution is more contracted than those in the other two solvents. These results are consistent with the compact packaging of mucin in the secretary granules that have elevated Ca2+ levels.

Rat uterine contraction by kallikrein and its dependence on uterine kininogen.

Smooth muscle responses to kallikrein (EC 3.4.21.8) are generally considered to result from kinin formation. In the present study, this premise was reexamined with respect to the isolated rat uterus. Rat submandibular gland kallikrein produced contractions of the rat uterus but the contractions disappeared after successive additions of the same dose of the enzyme to the preparation. Kallikrein-induced rat uterine contractions as well as bradykinin-induced contractions were enhanced by rat submandibular gland bradykinin potentiating factor. The incubation of kallikrein with rat uterine extract in the presence of a kininogen-depleted rat uterus produced kinin which elicited the uterine contraction. An extract from uterine horns previously depleted of kininogen was prepared. Incubation of this extract with kallikrein in a bath containing a kininogen-depleted rat uterus did not evoke uterine contraction. The incubation of four rat uterine horns with kallikrein in the presence of a uterine horn previously depleted of kininogen elicited contractions of the depleted uterus. These results suggest that the contraction produced by kallikrein involves kinin release from the uterus.

Molecular characterization of a functional cDNA encoding the rat substance P receptor.

Substance P is a member of the tachykinin peptide family and participates in the regulation of diverse biological processes. The polymerase chain reaction and conventional library screening were used to isolate a complementary DNA (cDNA) encoding the rat substance P receptor from brain and submandibular gland. By homology analysis, this receptor belongs to the G protein-coupled receptor superfamily. The receptor cDNA was expressed in a mammalian cell line and the ligand binding properties of the encoded receptor were pharmacologically defined by Scatchard analysis and tachykinin peptide displacement as those of a substance P receptor. The distribution of the messenger RNA for this receptor is highest in urinary bladder, submandibular gland, striatum, and spinal cord, which is consistent with the known distribution of substance P receptor binding sites. Thus, this receptor appears to mediate the primary actions of substance P in various brain regions and peripheral tissues.

The characterization of recombinant mouse glandular kallikreins from E. coli.

A system has been developed for the expression in E. coli of 12 of the 14 expressed mouse submandibular gland kallikreins as cassettes subcloned directly from cDNA. Using the epidermal growth factor binding protein (mGK-9) and the gamma-subunit of nerve growth factor (mGK-3), as test cases, mature processed forms, obtained as functionally active proteins, as well as various precursor forms, were isolated. The expression system described allows rapid isolation of kallikrein protein from corresponding cDNA with yields of approximately 1.0 mg of purified protein from 10 g of initial cell paste. This expression system will facilitate structure/function studies of the mouse glandular kallikrein gene family and help elucidate the regions of the mature proteins responsible for the diverse catalytic behavior and growth factor interactions observed in this family of proteins.

Differentiation of rat salivary glands by thermoanalytical analysis.

The water release from the sublingual, parotid and submandibular glands of male and female rats was analyzed by thermal analysis in order to detect the total water content and types. Different types of water, which are increasing from the sublingual to the parotid gland, were found and the relative distribution appeared to be a function of the bond energy of water to glandular components. In addition, evidence of a sexual dimorphism in the rat sublingual gland was demonstrated.

Expression of glutamine/glutamic acid-rich proteins in rat submandibular glands.

The expression of GRP transcripts was found to be highly specific to the rat submandibular gland. GRP cross-reactive species were detected in the saliva of both inbred and outbred rat strains. There was no evidence of GRP transcripts in RNA prepared from bovine, ovine, porcine or murine submandibular glands. Thus the GRPs differ from the family of PRPs that are expressed in several species. The restriction of GRP expression to the rat suggests a relatively recent origin for a functional GRP gene, presumably after rat-mouse divergence. The ontogeny of the relative steady-state levels of GRP transcripts was assessed by dot blot analysis. Maximal levels of RNA-encoding GRP were detected at 6 months; there was a significant age-related decline at both 12 and 18 months. There was, however, no significant age-related alteration in the size of transcripts which encode this protein family.

Immunohistochemical study of a rat membrane protein which induces a selective potassium permeation: its localization in the apical membrane portion of epithelial cells.

We previously reported a novel rat membrane protein that exhibits a voltage-dependent potassium channel activity on the basis of molecular cloning combined with an electrophysiological assay. This protein, termed IsK protein, is small and different from the conventional potassium channel proteins but induces selective permeation of potassium ions on its expression in Xenopus oocytes. In this investigation, we examined cellular localization of rat IsK protein by preparing three different types of antibody that specifically reacts with a distinct part of rat IsK protein. Immunohistochemical analysis using these antibody preparations demonstrated that rat IsK protein is confined to the apical membrane portion of epithelial cells in the proximal tubule of the kidney, the submandibular duct and the uterine endometrium. The observed tissue distribution of rat IsK protein was consistent with that of the IsK protein mRNA determined by blot hybridization analysis. In epithelial cells, the sodium, potassium-ATPase pump in the basolateral membrane generates a sodium gradient across the epithelial cell and allows sodium ions to enter the cell through the apical membrane. Thus, taking into account the cellular localization of the IsK protein, together with its electrophysiological properties, we discussed a possible function of the IsK protein, namely that this protein is involved in potassium permeation in the apical membrane of epithelial cells through the depolarizing effect of sodium entry.

In vitro radiolabeling of galactosyl and N-acetylgalactosaminyl moieties of glycoproteins with carbon-14.

The primary alcohol group on the carbon 6 of terminal galactosyl and N-acetylgalactosaminyl moieties of glycoproteins can be oxidized to an aldehyde by treatment with galactose oxidase. By reacting these aldehyde groups with 14C-labeled sodium cyanide, 14C-labeled cyanohydrin derivatives were obtained. Similarly, reduction of these aldehyde groups with tritiated sodium borohydride following standard procedures, yields 3H-labeled glycoproteins. 14C- and 3H-labeled derivatives of asialofetuin and asialo ovine submaxillary mucin with high specific radioactivities were prepared using these procedures. Mixtures containing microgram amounts of 14C- and 3H-labeled glycoproteins were subjected to column chromatography and gradient ultracentrifugation and the position of the individual glycoproteins was determined by simultaneous counting for 14C and 3H. These experiments demonstrate the usefulness of this approach for comparative analytical studies using biological specimens available in minute quantities.

Isolation and identification of a polypeptide in the Hsp 70 family that binds substance P.

During the course of an attempt to purify the substance P (SP) receptor from horse salivary glands by substance P-affinity chromatography, a polypeptide of Mr = 78,000 was isolated. The first fifteen amino acid residues at the amino terminus were determined and, unexpectedly, were found to be identical with the amino terminus of a glucose-regulated protein (GRP) of the same molecular weight, a protein that has been identified as a member of the heat shock protein family. This finding raises the intriguing possibility that SP may interact in vivo with GRPs and other members of the heat shock protein family and play a role in modulating their biological activities.

The amino-acid sequence of the double-headed proteinase inhibitor from badger (Meles meles) submandibular glands.

Badger submandibular glands contain a double-headed secretory proteinase inhibitor. Its amino acid sequence was determined. Extensive homologies were found between this inhibitor and the corresponding inhibitors of fox, dog, lion and cat in both domains. As in fox and dog inhibitor, the trypsin-inhibiting domain of badger inhibitor contains an Arg residue in the reactive site in contrast to a Lys residue in the inhibitors of lion and cat. Domains I and II of badger inhibitor are structurally related both to the sequenced inhibitors of fox, dog, lion and cat and to the sequenced monovalent secretory pancreatic trypsin inhibitors. The sequence of the badger inhibitor is N-terminally extended by four amino acids in comparison to fox and dog inhibitors and extended by eight amino acids in comparison to lion and cat inhibitors. Furthermore, the badger inhibitor is C-terminally extended by two amino acids in comparison to the lion inhibitor and by three amino acids in comparison to all other sequenced inhibitors.

Purification and properties of a Ca2+-independent sialic acid-binding lectin from human placenta with preferential affinity to O-acetylsialic acids.

A Ca2+-independent sialic acid-specific lectin from two developmental stages of human placenta was similarly purified to apparent homogeneity by DEAE-cellulose chromatography, affinity chromatography on bovine submaxillary mucin, and gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration disclosed a molecular mass of 53 kDa. The specificity of the lectin for O-acetylsialic acids was substantiated by the dependence of hemagglutination on the presence of acetylated sialic acids on the surface of mammalian erythrocytes of various sources, by hapten inhibition in hemagglutination assays with protease-treated rabbit erythrocytes and by hapten inhibition of binding of labeled N-acetylneuraminic acid-bovine serum albumin to the lectin in a solid-phase assay. Bovine and equine submaxillary mucins that contain 9(7,8)-O-acetyl and 4-O-acetylsialic acids were potent inhibitors in contrast to the non-acetylated sialic acids of ovine submaxillary mucin. Absence of inhibitory efficiency of other negatively charged substances like phosphorylated sugars, glucuronic acid, heparin, or oligodeoxynucleotides emphasized the importance of structural features instead of simple ionic interaction. In the presence of acetylation, the pattern of inhibition by gangliosides in the solid-phase assay indicated a preference to alpha-2,8- or alpha-2,6-linked sialic acids in comparison to alpha-2,3-linked moieties. Chemical modification of the lectin by group-specific reagents allowed to emphasize the role of primarily lysine residues, but also, although less pronounced, arginine, tryptophan, and carboxyl groups for ligand binding and/or maintenance of the active conformational state. Application of reagents, specific for histidine or tyrosine residues, failed to affect lectin activity.

Epidermal growth factor deficiency associated with diabetes mellitus.

The production of epidermal growth factor (EGF) in the submandibular gland and its circulating level were studied in diabetic mice. In genetically diabetic (C57BL/KsJ db/db) mice, EGF concentrations in the submandibular gland and plasma were reduced to 13% and 30% of the control levels, respectively. In streptozotocin-treated diabetic mice, they were reduced to 18% and 20% of controls, respectively, 5 weeks after the drug injection. Furthermore, levels of submandibular prepro-EGF mRNA in these diabetic mice were decreased almost in parallel with the glandular EGF concentrations, while there was no change in the levels of submandibular beta-actin mRNA and kidney prepro-EGF mRNA. In addition, histological examination of the submandibular glands indicated that the size of the granular convoluted tubules, which produce EGF, was substantially reduced in the diabetic mice. Insulin administration to streptozotocin-treated mice almost completely reversed the decrease in EGF content in the submandibular gland, substantially elevated the level of the glandular prepro-EGF mRNA and plasma EGF concentration, and increased the size of the granular convoluted tubules in the gland. These results indicate that EGF deficiency occurs in diabetes mellitus and that insulin may be important in maintaining the normal level of EGF in the submandibular gland and plasma.