A common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands.

A highly purified membrane preparation from rat parotid secretion granules has been used as a comparative probe to examine the extent of compositional overlap in granule membranes of three other exocrine secretory tissues--pancreatic, lacrimal, and submandibular--from several standpoints. First, indirect immunofluorescent studies using a polyclonal polyspecific anti-parotid granule membrane antiserum has indicated a selective staining of granule membrane profiles in all acinar cells of all tissues. Second, highly purified granule membrane subfractions have been isolated from each exocrine tissue; comparative two-dimensional (isoelectric focusing; SDS) PAGE of radioiodinated granule membranes has identified 10-15 polypeptides of identical pI and apparent molecular mass. These species are likely to be integral membrane components since they are not extracted by either saponin-sodium sulfate or sodium carbonate (pH 11.5) treatments, and they do not have counterparts in the granule content. Finally, the identity among selected parotid and pancreatic radioiodinated granule membrane polypeptides has been documented using two-dimensional peptide mapping of chymotryptic and tryptic digests. These findings clearly indicate that exocrine secretory granules, irrespective of the nature of stored secretion, comprise a type of vesicular carrier with a common (and probably refined) membrane composition. Conceivably, the polypeptides identified carry out general functions related to exocrine secretion.

Regulation of protein synthesis in the venom gland of viperid snakes.

Morphological changes in the venom gland of V. ammodytes were studied after the removal of the venom from the gland lumina (milking) It was found that the height of the secretory cells was changed during the secretory cycle. The patterns of the rough endoplasmic reticulum and of the Golgi complex were changed as well Milking induced an increased incorporation of [(14)C]amino acids into total and venom proteins In V ammodytes, during the first day after milking, 25% of the total counts in protein were precipitable by anti-venom serum, while at 8 days, 80% of the proteins synthesized were venom proteins At this stage, the incorporation was 10- and 20-fold that of unmilked glands for total and venom proteins, respectively. Venom was accumulated (secreted) in the gland lumina of V. ammodytes at a relatively high rate up to 2 wk after milking and leveled off afterwards. Intact glands and gland slices of V ammodytes and V palaestinae, taken from snakes a few days after milking, incorporated [(14)C]amino acids into proteins in vitro at a rate higher than that of unmilked glands. The activity of two exportable enzymes (phosphodiesterase and benzoyl arginyl ethyl esterase) was assayed in gland homogenates of V. ammodytes. It was found that 2-3 wk after milking, the intracellular level of these enzymes was up to 2-fold that of unmilked glands.

Composition of cellular membranes in the pancreas of the guinea pig. IV. Polyacrylamide gel electrophoresis and amino acid composition of membrane proteins.

TWO METHODS OF POLYACRYLAMIDE GEL ELECTROPHORESIS (THE ACID METHOD OF EYTAN AND OHAD AND THE NA DODECYLSULFATE (SDS) DISC METHOD OF MAIZEL) HAVE BEEN USED FOR ANALYZING THE PROTEINS OF GEL FRACTIONS ISOLATED FROM THE GUINEA PIG PANCREATIC EXOCRINE CELLS AND IN PARTICULAR THE PROTEINS BOUND TO THE MEMBRANES INVOLVED IN THE SYNTHESIS, INTRACELLULAR TRANSPORT, AND DISCHARGE OF SECRETORY ENZYMES: rough (RM) and smooth microsome (SM) membranes, zymogen granule (ZG) membranes, and plasma membranes (PM). Since in the two systems the electrophoretic mobility of proteins depends on different factors (size, shape, and net charge of molecules in the acid system; size only in the SDS system) a deeper insight into the protein composition of the fractions could be obtained. The gel patterns of RM, SM, and ZG membranes turned out to be accounted for mainly by segregated secretory enzymes (in rough microsomes also by ribosome proteins) and thus were found to share most of the bands. In contrast, with highly purified membrane fractions different patterns were obtained: RM and SM membrane proteins turn out to contain a large number of different proteins with molecular weights varying between approximately 150,000 and 15,000 daltons. The pattern of ZG membranes was greatly different in the two systems: only two bands were separated by the acid method and as many as 23 by the SDS method. PM gave a rather complex pattern in either system. Both ZG membranes and PM were found to contain a large proportion of low molecular weight proteins. Nothing appears in common between the proteins of SM membranes (primarily of Golgi origin) and those of ZG membranes, while the latter and PM exhibit a certain degree of similarity. By amino acid analysis we found only slight differences: relative to the other fractions: RM membranes were higher in basic amino acids and ZG membranes contained a larger amount of methionine. Taken together with recent data on lipid composition and enzyme activities of the same fractions, these results indicate that the membranes of the pancreatic exocrine cells are chemically and functionally distinct, and hence do not mix randomly with one another during the transport of secretory products.

Soluble and membrane-bound muscarinic acetylcholine receptors.

Three muscarinic acetylcholine receptor subtypes may be defined on the basis of functional and binding studies using selective antagonists. The subtypes may be solubilized in a stable form in digitonin. In solution, the subclasses still exhibit different structure-binding relationships but these have been perturbed by solubilization. The binding of the selective antagonist, pirenzepine, to the purified cortical receptor is complex and similar to that found in membranes. The muscarinic receptor subclasses thus appear to be different molecular entities. Possible explanations for the molecular heterogeneity are discussed. It has also been possible to solubilize receptor-GTP binding protein complexes which have higher sedimentation coefficients (13.4 S) than the apparently monomeric receptor (11.6 S).

Structural studies on RNA from Bombyx mori L. I. Nucleoside composition of enriched tRNA species from the posterior silkgland purified by coutercurrent distribution.

A large scale fractionation of tRNA from the posterior silkgland of the silkworm Bombyx mori L. by countercurrent distribution is described. One single 1,500 transfer distribution carried out with Phosphate buffer-Fromamide-Isopropanol (PFI) solvent system yields highly enriched isoaccepting species with increasing mobility order: tRNA1Gly, tRNA1-2Ala, tRNATyr, tRNA2Gly, tRNA1Ser and tRNA2Ser with 75%, 70%, 90%, 60%, 60%, and 90% purities respectively. Nucleosides fingerprint analysis of each iso-tRNA species confirms the anticodon structures previously suggested for tRNA2Ala (IGC), tRNA2bGly (U-CC) (U-CC) and tRNA2bSer (IGA). Twenty two minor nucleosides, three of them with unknown structure, have been detected. They are: m5C in tRNA1Gly, m1I in all tRNAAla species, polar A and U called X in tRNATyr, polar U derivative in tRNAGly2, mt6A in tRNASer1 and i6A tRNA2Ser. Both tRNASer species have m3C and ac7C. We do not detect Q, Y and thiol derivatives. The elution characteristics of silkgland tRNA species may be expressed in a semilogarithmic diagram where log K (K is the partition coefficient) is related to the base ratio A/Y) and the coding properties. The distribution pattern of silkgland tRNAs has been compared with that of Yeast and Rat liver tRNAs fractionated by countercurrent distribution with the PFI and PMB (Potassium phosphate buffer, 2-methoxy ethanol, 2-butoxy ethanol) solvent systems.

Quantitative data on the Bombyx mori L. silkworm: a review.

This paper summarizes a variety of quantitative data on the silkworm Bombyx mori, collected in the literature, to help building models on silk gland differentiation. The properites of the silk gland and their changes especially during the last larval instar have been reviewed (size, DNA, RNA amino acids, enzymes). The components of the silk (fibroin and sericin) are also studied (molecular weight, composition). Thus translation and transcription rates have been estimated. The relevant data on the fat body and the haemolymph are also given, as well as some characteristics of the oocyte/egg system.

Immunological characterization of a membrane glycoprotein of chromaffin granules: its presence in endocrine and exocrine tissues.

A glycoprotein was isolated from detergent solubilized membranes of bovine chromaffin granules by high-performance liquid chromatography. Specific antisera raised against this glycoprotein reacted in one- and two-dimensional immunoblots with a heterogeneous component with a pI of 4.2-4.7 and Mr 100,000. The antiserum against bovine glycoprotein II cross-reacted with an analogous component in several species. The specific localization of glycoprotein II in chromaffin granules was established by density gradient centrifugation followed by immunoblotting. The antiserum, as shown by one- and two-dimensional immunoblotting, reacted with an analogous antigen in the posterior pituitary, in endocrine (anterior pituitary, parathyroid gland) and exocrine (parotid gland, pancreas) organs. In the pancreas the protein reacting with the antiserum was found in the membranes of zymogen granules. The results demonstrate for the first time that secretory vesicles of endocrine and exocrine tissues have at least one common antigen, i.e. the glycoprotein II. It seems likely that this protein is involved in a basic function common to all secretory vesicles.

Quantitative histochemistry of mucosubstance in tracheal epithelium of the macaque monkey.

Experimentally applied irritants and chronic respiratory diseases appear to alter the amount and composition of secretory cell product in surface epithelium and submucosal glands of pulmonary airways. Previous methods used to quantify these changes have been very time-consuming or have not measured the same components of the airway wall. The present study describes a rapid, reproducible, and standardized automated method for quantifying secretory products. The tracheas from eight macaque monkeys were fixed with glutaraldehyde-paraformaldehyde, embedded in glycol methacrylate, serially sectioned at 2 microns, and histochemically stained to demonstrate neutral, sialylated, and sulfated mucosubstances in the cartilaginous, intercartilaginous, and membranous regions of both proximal and distal trachea. Volume densities were determined using an image analyzer and are expressed as volume of stained mucosubstance per unit surface area of epithelial basal lamina. Comparison of the automated method to manual point counting and evaluation of internal variance showed that the automated method had a twelve-fold increase in efficiency with no significant differences in measurements. After weighting the values of each region according to their anatomical contribution, the total secretory product (TSP) for the entire trachea was determined. Periodate-reactive acid material predominated (73%) in luminal surface epithelium, and neutral material predominated (78%) in submucosal glands. Surface epithelium contained 66% of the TSP. The greater contribution by surface epithelium and predominance of acid mucins there resulted in a TSP from the trachea that consisted of 59% acid material (most of which was sulfated) and 41% neutral material. The method proved to be a valid, reproducible, and rapid technique for evaluating variability in abundance of mucosubstances within airway epithelium.

Immunohistochemical localization of smooth muscle myosin in normal human tissues.

Immunohistochemical localization of smooth muscle myosin, an immunologically distinct contractile protein, was achieved using rabbit anti-human uterine smooth muscle myosin antibodies. In immunodiffusion studies and in cryostat sections, these antibodies were highly specific and reacted with smooth muscle myosin but not with platelet, skeletal muscle, or cardiac muscle myosin. To evaluate comprehensively the structural profile of smooth muscle elements in normal human tissues, an indirect immunoperoxidase technique (peroxidase-antiperoxidase) was applied to a wide variety of specimens. Parallel studies comparing cryostat sections with fixed (10% formalin, B5, Bouin's, or Zenker's solution) paraffin-embedded tissues revealed optimal immunoreactivity, sensitivity, and specificity of staining for smooth muscle myosin using frozen tissues. Strong immunoreactivity was present in muscular tissues such as blood vessels and the muscularis of gastrointestinal and genitourinary tracts. Distinct delineation of smooth muscle elements, including individual smooth muscle cells, and their specific patterns of alignment and organization, were observed, e.g., cells comprising the muscularis mucosae and extending into the lamina propria of the gastrointestinal tract, and myoepithelial cells of skin, exocrine glands, and breast. This method provides excellent morphologic preservation and readily permits unambiguous identification of individual cells containing smooth muscle myosin.

Adenosine deaminase complexing proteins are localized in exocrine glands of the rabbit.

Adenosine deaminase complexing proteins have been localized in four exocrine glands of the rabbit by immunoperoxidase staining employing affinity-purified goat anti-rabbit complexing protein immunoglobulin as the primary antibody. In pancreatic acinar cells and in serous cells of Brunner glands (duodenal glands), staining was concentrated in granular appearing deposits between the nucleus and cell apex. Bile canaliculi, components of the exocrine liver, were also positive for complexing protein. In submaxillary glands, staining was localized in serous demilunes and striated ducts. In each instance staining was blocked by preincubating the primary antibody with complexing protein purified from rabbit kidney.

Similarities and differences among neuroendocrine, exocrine, and endocytic vesicles.

Secretory and endocytic vesicles have analogous functions as cyclic carriers between specific cellular compartments. The centrifugally functioning secretory system operates from the Golgi complex, whereas the centripetally functioning endocytic system operates from the cell surface. Further, within polarized epithelial cells the export traffic can be directed to a distinct plasmalemmal domain which distinguishes exocrine from endocrine secretion and import traffic can be directed transcellularly. These shuttle operations involve a special class of lipid-rich, protein-poor membranes that appear to use an inwardly directed H+-translocase activity to varying extents for pH-dependent sorting and for accumulation and concentration of transported molecules. Comparative analyses of purified membrane preparations from exocrine and endocrine sources identify compositional overlap between different types of shuttle membrane. However, the structural elements that specify a particular origin or destination for a given carrier or determine function in storage and stimulus-dependent shuttling remain unknown.

An investigation of venom secretion by the venom gland cells of the carpet viper (Echis carinatus).

The indirect immunofluorescent antibody technique was applied to the study of Echis carinatus pyramidum venom antigens in venom gland tissue using semi-thin frozen sections. A total of four rabbit antisera, two monoclonal antibodies active against E. carinatus venom, two monoclonal antibodies active against the rodent malaria parasite, Plasmodium chabaudi, and two monoclonal antibodies active against the human malaria parasite, Plasmodium falciparum, were investigated. The results of this study suggest that each secretory cell within the main part of the gland produces all the venom constituents. The resultant venom is therefore considered to be produced as a single package by each individual secretory cell. The different constituents of the venom studied are not produced at the same time or at the same rate throughout the secretory cycle, some being produced at the beginning and others at a later stage.

Effect of venom gland extracts of the South American brown spider, Loxosceles laeta, on in vitro protein synthesis.

There was no effect of the venom extract on incorporation of phenylalanine into protein from phenylalanyl-tRNA. In contrast, the esterification of amino acids with tRNA was inhibited by the venom extracts in a dose-dependent manner, with an apparent preservation of the covalent structure of aminoacyl-tRNA.

Hamster nasal glands: their structure, sialic acid content, and vulnerability to actinomycin D.

This study examines the structure of mucosal glands in the walls of the hamster maxillary recess, compares the histochemical appearance of nasal glands to their sialic acid content, and determines the vulnerability of nasal glands to actinomycin toxicity. Observations were made on plastic-embedded tissue with light and transmission electron microscopes. Determinations of total sialic acid in mucosal samples were conducted with thiobarbituric acid. Experimental hamsters were administered 0.2 micrograms of actinomycin D (IP)/gm body weight/day for five days. Types of granules present in the later nasal gland (LNG) and glands of the maxillary recess (MRGs) include: 2.0 micrometers lightly basophilic, lightly electron-dense granules and 1.5 micrometers strongly basophilic, electron-dense granules in the same acinar cell type in both the LNG and MRGs; 1.5 micrometers metachromatic granules in some acinar cells of the LNG; 1.0 micrometer moderately electron-dense granules in cells of MRG ducts; and 0.7 micrometers electron-dense granules in cells of LNG intercalated ducts. Acid glycoproteins, demonstrable by histochemistry, are present in the LNG but not in the MRGs. However, the total sialic acid content of tissues from MRG tissue is greater than that of other tissues measured. A minor number of LNG acini, those with metachromatic granules, have branching basal cytoplasmic projections. Many dark cells are present in striated ducts of the LNG. Histological alteration due to actinomycin-D toxicity, conspicuous in parotid salivary parenchyma, is greater in MRG than in LNG tissue.

Tracheal submucosal gland development in the rhesus monkey, Macaca mulatta: ultrastructure and histochemistry.

The submucosal glands are thought to be the primary source of the mucus overlying the primate trachea and conducting airways. This study characterizes the development of submucosal glands in the trachea of the rhesus monkey. Tracheas from 46 age-dated fetal, 8 postnatal and 3 adult rhesus were fixed in glutaraldehyde/paraformaldehyde and slices processed for electron microscopy. The earliest (70 days gestational age (DGA)) indication of gland development was the projection of a group of closely packed electron lucent cells with few organelles and small pockets of glycogen into the submucosa. This configuration was observed up to 110 DGA. In fetuses younger than 87 DGA it was present almost exclusively over cartilaginous areas. Between 80 and 140 DGA, a cylinder of electron lucent cells projected into the submucosal connective tissue perpendicular to the surface. In fetuses younger than 100 DGA, it was restricted to cartilaginous areas. By 90 DGA, some glycogen containing cells in proximal regions contained apical cored granules. By 106 DGA, cells in proximal areas contained apical electron lucent granules. More distal cells had abundant GER and electron dense granules. The most distal cells resembled the undifferentiated cells at younger ages. Ciliated cells were present in the most proximal portions of glands at 120 DGA. This glandular organization was found in older animals, including adults, with the following changes: abundance of proximal cells with electron lucent granules increased; abundance of distal cells with electron dense granules increased; and abundance of distal cells with abundant glycogen and few organelles decreased. We conclude that submucosal gland development in the rhesus monkey: is primarily a prenatal process; occurs first over cartilage; continues into the postnatal period; and involves secretory cell maturation in a proximal to distal sequence with mucous cells differentiating before serous cells.

Identification and quantitation of arachidonic-acid metabolic products in rabbit, rat and human saliva.

Arachidonic-acid metabolites were identified in salivary secretions of rabbits, rats and man and in homogenates of rabbit parotid and submandibular glands. The following levels were determined in mixed saliva of healthy volunteers: PGE2, 0.65 +/- 0.8 ng/ml; PGF2 alpha, 0.41 +/- 0.06 ng/ml; serologically active hydroxyeicosatetraenoic acids (HETE) 319.6 +/- 36.6 ng/ml; serologically active 6-sulphidopeptide-containing leukotrienes 1.72 +/- 0.21 ng/ml (all values mean +/- SEM). In all instances, HETE markedly predominated over all other arachidonic-acid metabolites.

Activities of novel polyhydroxylated cardiotonic steroids purified from nuchal glands of the snake, Rhabdophis tigrinus.

Seven novel polyhydroxylated steroids were isolated from the nucho-dorsal glands of the snake, Rhabdophis tigrinus. Biological activities of these steroids in inhibiting (Na+ + K+)ATPase and in producing positive inotropic action were examined in comparison with those of ouabain and gamabufotalin. Gamabufotalin was approximately 10 times more potent than ouabain in inhibiting (Na+ + K+)ATPase. Two compounds, compounds III and XIII, of the seven, produced nearly equipotent enzyme inhibitory activity to ouabain. The activity of the remaining five was relatively low among the compounds tested. All compounds exhibited more or less positive inotropic action in the papillary muscle preparations. The ranking order of the potency was: gamabufotalin greater than ouabain and compound IV greater than compound III and XIII greater than compound I, II, XII and XIV.

Ultrastructural and cytochemical study on the enzyme gland of the foot of a mollusc.

The enzyme gland of the foot of the mussel Mytilus has been so far considered a gland producing and exporting a phenol oxidase catalysing the general tanning processes of byssus threads. In contrast, the present study shows that this gland produces mainly secretory granules which form the cortical layers of byssus threads. Cytochemical methods at the ultrastructural level (phosphotungstic acid at low pH, silver methenamine, periodic acid-thiosemicarbazide-silver proteinate, silver methenamine for sulphur-rich proteins demonstration) and enzyme digestion tests (pepsin, trypsin, alpha-chymotrypsin) indicate that secretory granules contain glycoproteins rich in sulphydryl groups and in aromatic amino acids. The cytochemical demonstration of phenol oxidase shows that enzyme activity is present in Golgi complex, whereas it is absent in secretory granules. For this reason, phenol oxidase does not seem to be exported and utilized for tanning of byssus threads, but it might rather be involved in the elaboration and tanning of the content of the secretory granules in the enzyme gland itself.

Occurrence of 7-dehydrocholesterol in the uropygial gland of domestic fowls.

Histochemical studies on the uropygial gland of domestic fowls have shown the presence of sterols (among which cholesterol and its esters) in the lipidic fraction of the gland secret. beta-Glucuronidase activity beside A5 3beta- and 17beta-hydroxysteroid dehydrogenase activities suggests that uropygial gland might be involved in sterols metabolism. By thin layer chromatography cholesterol and 7-dehydrocholesterol can be separated from the uropygial extracts and these compounds can be identified in gas liquid chromatography.

[Histochemical studies of mucopolysaccharides in the urethral glands of newborns (glands of Littre) (author's transl)]. / Etude histochimique des mucopolysaccharides des glandes uréthrales du nouveau-né (glandes de Littre)

Histochemical study of urethral glands (Littre's glands) at birth shows, from that time, a notable secretion of mucopolysaccharides. It appears that the properties of the prosecretion are, in part, different from those described in the adult. In fact, in addition to the acid mucins which loose their affinity for Alcian dyes after digestion by neuraminidase and acid hydrolysis, there is an important elaboration of neutral mucosubstances which reacts positively with PAS even if the latter is combined with Alican Blue staining.