scholarly journals Monoclonal antibodies against rat saliva and salivary gland antigens.

1985 ◽  
Vol 33 (3) ◽  
pp. 209-218 ◽  
Author(s):  
T Barka ◽  
E W Gresik ◽  
H van der Noen
Keyword(s):  
Submandibular Gland ◽  
Solid Phase ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Adult Rats ◽  
Saliva Secretion ◽  
Old Rats ◽  
Hybridoma Cell Lines ◽  
Ascites Tumors ◽  
Submandibular Saliva

Hybridomas were produced by the fusion of NS1 myeloma cells with spleen cells of a BALB/c mouse immunized with rat submandibular saliva. Growth of hybridomas was evident in 60/96 wells, and colonies secreting antibodies against saliva components were identified in 20 wells by using a solid phase enzyme-linked immunoassay. Cloning of cells from 12 wells yielded originally 43 hybridoma cell lines secreting anti-saliva antibodies. After recloning, one hybridoma (4Cl3) was selected for further studies. The hybridoma (4Cl3) cells were grown as ascites tumors, and the antibodies were purified from the ascitic fluid by diethylaminoethyl Affi-gel Blue chromatography. The purified antibody (MA4), immunoglobulin G1, immunoprecipitated a 39K dalton protein from submandibular saliva, and also reacted with a protein of the same electrophoretic mobility on immunoblots. From extracts of submandibular gland slices, incubated with [3H]leucine, the antibody again immunoprecipitated a 39K protein, indicating that this protein is synthesized in the gland. MA4 was used for immunocytochemical stainings of submandibular glands of rats of different ages. In general, immunostaining was seen only in acinar cells. Thus, there was no staining in the glands of 1-day-old rats that lack differentiated acinar cells. In the glands of 1- to 4-week-old rats the number of immunoreactive cells and the extent of immunostaining paralleled the differentiation of the acinar cells. In the glands of adult rats a uniform staining of the secretory granules of the acinar cells was observed. The immunoreactive 39K protein seemed to be restricted to the acinar cells in the submandibular gland; there was no immunostaining in the parotid, sublingual, or lingual salivary glands, or in the pancreas, colon, and duodenum. Stimulation of saliva secretion by isoproterenol resulted in a virtual depletion of the antigen from the acinar cells. These results indicate the feasibility of producing mouse hybridomas that secrete antibodies against rat saliva components. The monoclonal antibody at hand will be useful in analyzing the differentiation of the acinar cells, and the factors that influence this differentiation process.

Immunocytochemical localization of amylase in the parotid gland of developing and adult rats.

1981 ◽  
Vol 29 (10) ◽  
pp. 1189-1195 ◽  
Author(s):  
T Tanaka ◽  
E W Gresik ◽  
T Barka
Keyword(s):  
Secretory Granules ◽  
Protein A ◽  
Acinar Cells ◽  
Gold Colloid ◽  
Parotid Glands ◽  
Adult Rats ◽  
Old Rats ◽  
Rat Parotid ◽  
Unlabeled Antibody ◽  
Number Of Cells

An antiserum against purified rat parotid amylase was used to localize the protein in parotid glands of developing and adult rats. The unlabeled antibody peroxidase-antiperoxidase method and the protein A-gold colloid technique were used at the light and electron microscope levels, respectively. Immunoreactive amylase was detected in a few scattered cells in the glands of 2-day-old rats. During the following days the number of cells stained immunocytochemically for amylase increased rapidly; at 15 days of age all acinar cells revealed amylase, but the intensity of immunostaining varied from cell to cell. Electron microscopically, amylase was localized in the secretory granules, and by using a more concentrated antiserum, in the rough endoplasmic reticulum and Golgi complex. At early stages of development the acinar cells contained fewer and smaller secretory granules than in adult animals; the gold particles indicative of amylase were randomly distributed over the secretory granules. In the glands of adult rats, amylase was distributed inhomogeneously within the secretory granules. In the majority of secretory granules gold colloid particles were located over the electron-dense portions of the granules. However, secretory granules in which an amylase-rich shell surrounded an amylase-poor or amylase-negative "core" were not infrequent.

scholarly journals LOCALIZATION OF PEROXIDASE ACTIVITY IN THE DEVELOPING SUBMANDIBULAR GLAND OF NORMAL AND ISOPROTERENOL-TREATED RATS

1972 ◽  
Vol 20 (11) ◽  
pp. 855-872 ◽  
Author(s):  
SHOHEI YAMASHINA ◽  
TIBOR BARKA
Keyword(s):  
Endoplasmic Reticulum ◽  
Enzyme Activity ◽  
Submandibular Gland ◽  
Peroxidase Activity ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Chronic Administration ◽  
Secretory Material ◽  
Old Rats ◽  
Tubule Cells

The localization of peroxidase activity was studied in the developing submandibular gland of normal and isoproterenol-treated rats using a diaminobenzidine-H2O2 method. During postnatal development peroxidase activity was localized in the proacinar and acinar cells. The proacinar cells were characterized by the presence of polymorphic secretory granules that gave a strong peroxidase reaction, particularly in the glands of 1-day-old rats. In addition to the secretory granules, enzyme activity was demonstrated in the rough endoplasmic reticulum and nuclear envelope. The terminal tubule cells and duct cells were devoid of peroxidase activity. The secretory granules in the fully developed acinar cells revealed little or no enzyme activity. Isoproterenol stimulated the secretion of the peroxidase-positive granules from the proacinar cells. The stimulation was followed by a reaccumulation of peroxidase-positive secretory material. During this process enzyme activity was demonstrable in the Golgi complex. Isoproterenol had no effect on the terminal tubule cells. A less effective depletion of the secretory granules from the proacinar cells was seen after pilocarpine administration. Chronic administration of isoproterenol to 5-day-old rats led to an acceleration of the differentiation of acinar cells and to a hypertrophy of the gland, without significant change in the localization of peroxidase activity.

scholarly journals Immunoelectron microscopy of carbonic anhydrase isozyme VI in rat submandibular gland: comparison with isozymes I and II.

1992 ◽  
Vol 40 (6) ◽  
pp. 807-817 ◽  
Author(s):  
Y Ogawa ◽  
C K Chang ◽  
H Kuwahara ◽  
S S Hong ◽  
S Toyosawa ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Submandibular Gland ◽  
Secretory Granules ◽  
Polyacrylamide Gel Electrophoresis ◽  
Acinar Cells ◽  
Enzyme Digestion ◽  
Immunoperoxidase Technique ◽  
Carbonic Anhydrase Isozyme ◽  
Indirect Immunoperoxidase ◽  
Rat Submandibular Gland

Carbonic anhydrase (CA) was purified from the saliva of pilocarpine-treated rats by inhibitor-affinity chromatography, and its localization in the rat submandibular gland was studied by the indirect immunoperoxidase technique using a monoclonal antibody (MAb) raised against the enzyme. SDS-polyacrylamide gel electrophoresis of the CA VI gave three bands of 33, 39, and 42 KD. Enzyme digestion experiment showed that the 42 KD molecule was degraded into the 39 KD molecule and the 39 KD molecule into the 33 KD molecule. The cleavage of the 42 KD molecule was independent and that of the 39 KD molecule was dependent on endo-beta-N-acetylglucosaminidase F. The 42 KD molecule was detected in the CA purified from the pilocarpine-treated but not the untreated salivary gland. The MAb recognized all the three components of the enzyme. Immunostaining for CA VI was seen in the cytosol and secretory granules of serous acinar cells and in the duct luminal contents. Staining specific for erythrocyte CA (CA I and CA II) was observed in the cytosol of the epithelial cells of granular, striated, and excretory ducts. Among these duct cells, the agranular varieties in the granular and excretory ducts were essentially devoid of the immunoreactivity.

scholarly journals Immunoelectron microscopy of carbonic anhydrase isozyme VI in human submandibular gland: comparison with isozymes I and II.

1993 ◽  
Vol 41 (3) ◽  
pp. 343-351 ◽  
Author(s):  
Y Ogawa ◽  
S S Hong ◽  
S Toyosawa ◽  
H Kuwahara ◽  
M Shimazaki ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Carbonic Anhydrase ◽  
Submandibular Gland ◽  
Polyclonal Antibodies ◽  
Secretory Granules ◽  
Polyacrylamide Gel Electrophoresis ◽  
Acinar Cells ◽  
Protein Band ◽  
Immunoperoxidase Technique ◽  
Dot Blotting

Carbonic anhydrase VI (CA VI) was purified from human saliva by inhibitor-affinity chromatography, and its distribution was studied in human submandibular gland by the indirect immunoperoxidase technique with a rabbit polyclonal antibody raised against the isozyme. Polyclonal antibodies to human CA I and CA II purified from erythrocytes were also raised and used for immunostaining. SDS-polyacrylamide gel electrophoresis of the purified isozymes revealed a single protein band (CA VI, 42 KD; CA I and CA II, 30 KD). Antibody raised against CA VI did not crossreact with CA I or CA II either by Western or by dot-blotting. However, antibodies against CA I and CA II showed slight crossreaction with each other's antigen by dot-blotting. In a Western blot of purified submandibular gland CA, antibody to CA VI stained the 42 and 30 KD bands, and antibodies to CA I and CA II stained the 30 KD band. The 42 KD but not the 30 KD molecule was cleaved by endo-beta-N-acetylglucosaminidase F, indicating that the former contains N-linked oligosaccharides. Immunostaining for CA VI was seen in the secretory granules and cytosol of serous acinar cells and in the duct luminal contents. Staining specific for CA II was observed in the cytosol of serous acinar and duct epithelial cells. Antibody to CA I reacted only with the walls of small blood vessels. These results suggest that (a) serous acinar cells secrete 42 KD CA VI which functions in the oral cavity and that (b) serous acinar and duct epithelial cells possess cytosolic CA (30 KD CA VI and CA II) which functions in situ.

scholarly journals Ultrastructural localization of salivary acidic proline-rich proteins from Macaca fascicularis.

1982 ◽  
Vol 30 (3) ◽  
pp. 274-278 ◽  
Author(s):  
E E Kousvelari ◽  
F G Oppenheim ◽  
L S Cutler
Keyword(s):  
Golgi Apparatus ◽  
Submandibular Gland ◽  
Subcellular Distribution ◽  
Macaca Fascicularis ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Ultrastructural Localization ◽  
Submandibular Glands

Using an indirect immunoferritin method, the subcellular distribution of acidic proline-rich proteins (MPRP) in the acinar cells of the macaque parotid and submandibular glands was defined. MPRP was found in the "spherule" substructure of the secretory granules as well as in specific Golgi transfer vesicles and in vesicles budding from the Golgi apparatus. The data suggest that the acidic proline-rich proteins of the primate Macaca fascicularis are synthesized and packaged by conventional exocrine mechanisms. In addition, these proteins appear to be packaged and transferred to the secretory granules of parotid and submandibular gland acinar cells as discrete aggregates that remain as a separate "spherule" area in the secretory granules.

scholarly journals Differential expression of proline-rich proteins in rabbit salivary glands.

1992 ◽  
Vol 40 (9) ◽  
pp. 1393-1404 ◽  
Author(s):  
F D Ferreira ◽  
R Robinson ◽  
A R Hand ◽  
A Bennick
Keyword(s):  
Parotid Gland ◽  
Salivary Glands ◽  
Submandibular Gland ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Ultrastructural Localization ◽  
Northern Blotting ◽  
In Vitro Translation ◽  
Specific Expression

Salivary glands synthesize and secrete an unusual family of proline-rich proteins (PRPs) that can be broadly divided into acidic and basic PRPs. We studied the tissue-specific expression of these proteins in rabbits, using antibodies to rabbit acidic and basic PRPs as well as antibodies and cDNA probes to human PRPs. By immunoblotting, in vitro translation, and Northern blotting, basic PRPs could be readily detected in the parotid gland but were absent in other salivary glands. In contrast, synthesis in vitro of acidic PRPs was detected in parotid, sublingual, and submandibular glands. Ultrastructural localization with immunogold showed heavy labeling with antibodies to acidic PRPs of secretory granules of parotid acinar cells and sublingual serous demilune cells. Less intense labeling occurred in the seromucous acinar cells of the submandibular gland. With antibodies to basic PRPs, the labeling of the parotid gland was similar to that observed with antibodies to acidic PRPs, but there was only weak labeling of granules of a few sublingual demilune cells, and no labeling of the submandibular gland. These results demonstrate a variable pattern of distribution of acidic and basic PRPs in rabbit salivary glands. These animals are therefore well suited for study of differential tissue expression of PRPs.

Further Studies on the Ultrastructure of Harderian Gland in Adult Rats

1974 ◽  
Vol 32 ◽  
pp. 288-289
Author(s):  
Alfredo Feria-Velasco ◽  
Guadalupe Tapia-Arizmendi
Keyword(s):  
Fine Structure ◽  
Domestic Fowl ◽  
Animal Species ◽  
Acinar Cells ◽  
Harderian Gland ◽  
Myoepithelial Cells ◽  
The Other ◽  
Albino Rats ◽  
Adult Rats ◽  
Cell Components

The fine structure of the Harderian gland has been described in some animal species (hamster, rabbit, mouse, domestic fowl and albino rats). There are only two reports in the literature dealing on the ultrastructure of rat Harderian gland in adult animals. In one of them the author describes the myoepithelial cells in methacrylate-embbeded tissue, and the other deals with the maturation of the acinar cells and the formation of the secretory droplets. The aim of the present work is to analize the relationships among the acinar cell components and to describe the two types of cells located at the perifery of the acini.

TNF-α Suppresses Autophagic Flux in Acinar Cells in IgG4-Related Sialadenitis

2019 ◽  
Vol 98 (12) ◽  
pp. 1386-1396 ◽  
Author(s):  
X. Hong ◽  
S.N. Min ◽  
Y.Y. Zhang ◽  
Y.T. Lin ◽  
F. Wang ◽  
...  
Keyword(s):  
Acinar Cell ◽  
Cell Injury ◽  
Ex Vivo ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Autophagic Flux ◽  
C6 Cells ◽  
Tnf Α ◽  
Α Level

IgG4-related sialadenitis (IgG4-RS) is a newly recognized immune-mediated systemic fibroinflammatory disease that affects salivary glands and leads to hyposalivation. Tumor necrosis factor–α (TNF-α) is a critical proinflammatory cytokine involved in several salivary gland disorders, but its role and mechanism regarding acinar cell injury in IgG4-RS are unknown. Here, we found that TNF-α level was significantly increased in serum and submandibular gland (SMG) of patients and that serum TNF-α level was negatively correlated with saliva flow rate. Ultrastructural observations of IgG4-RS SMGs revealed accumulation of large autophagic vacuoles, as well as dense fibrous bundles, decreased secretory granules, widened intercellular spaces, swollen mitochondria, and expanded endoplasmic reticulum. Expression levels of LC3 and p62 were both increased in patients’ SMGs. TNF-α treatment led to elevated levels of LC3II and p62 in both SMG-C6 cells and cultured human SMG tissues but did not further increase their levels when combined with bafilomycin A1 treatment. Moreover, transfection of Ad-mCherry-GFP-LC3B in SMG-C6 cells confirmed the suppression of autophagic flux after TNF-α treatment. Immunofluorescence imaging revealed that costaining of LC3 and the lysosomal marker LAMP2 was significantly decreased in patients, TNF-α–treated SMG-C6 cells, and cultured human SMGs, indicating a reduction in autophagosome-lysosome fusion. Furthermore, the ratio of pro/mature cathepsin D was elevated in vivo, ex vivo, and in vitro. TNF-α also appeared to induce abnormal acidification of lysosomes in acinar cells, as assessed by lysosomal pH and LysoTracker DND-26 fluorescence intensity. In addition, TNF-α treatment induced transcription factor EB (TFEB) redistribution in SMG-C6 cells, which was consistent with the changes observed in IgG4-RS patients. TNF-α increased the phosphorylation of extracellular signal–regulated kinase (ERK) 1/2, and inhibition of ERK1/2 by U0126 reversed TNF-α–induced TFEB redistribution, lysosomal dysfunction, and autophagic flux suppression. These findings suggest that TNF-α is a key cytokine related to acinar cell injury in IgG4-RS through ERK1/2-mediated autophagic flux suppression.

scholarly journals Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis

1994 ◽  
Vol 124 (1) ◽  
pp. 43-53 ◽  
Author(s):  
BP Jena ◽  
FD Gumkowski ◽  
EM Konieczko ◽  
GF von Mollard ◽  
R Jahn ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Binding Protein ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Apical Plasma Membrane ◽  
Gtp Binding Protein ◽  
Regulated Exocytosis ◽  
Gtp Binding

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3-like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.

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