Receptors on airway gland cells.

Airway submucosal glands are by volume the most important source of macromolecules in airway secretions. These secretions, containing gel-forming mucins, antibacterial proteins, and antiproteases, comprise the major defensive barrier protecting the host against airborne pathogens. The identification of the mechanisms regulating secretion from the submucosal glands is key to understanding the genesis of this barrier and how it is altered by disease processes. Using a variety of methods, we and others have identified on the gland cells of several species receptors specific for ACh, norepinephrine, substance P, VIP, PGE1, PGE2, PGA1, PGD2, histamine and bradykinin. These receptors all participate in modulating the secretory activity of the airway submucosal glands. Studies of homogeneous cultures of bovine airway serous cells have yielded detailed information regarding the beta-adrenergic receptor on these cells. Using radioligand binding techniques, we found evidence for the presence of a single high affinity beta receptor of beta-2 subtype. Occupancy of this receptor by isoproterenol causes an elevation in the concentration of intracellular cAMP, which in turn stimulates the phosphorylation of a subset of cytoplasmic and membrane proteins. Based on the kinetics and pharmacology of these effects, it is likely that cAMP functions as a second messenger in the serous cell secretory pathway, probably acting through protein kinases. Current efforts are directed at identification of those phosphoproteins whose phosphorylation and dephosphorylation times are consistent with their possible roles in secretion.

Histology, ultrastructure and carbohydrate histochemistry of pig carpal glands.

The morphological differences between two types of secretory cells (clear and dark) from pig carpal glands were examined. The main difference is the presence in the dark cells of secretory granules of moderate electron density, made up of acidic and neutral glycoproteins. The possible functional purposes of the carpal glands and of glycogen present in both cell types are also discussed.

Histochemistry of complex carbohydrates in the horse duodenal gland.

Complex carbohydrates were examined in glandular cells of the horse duodenal gland by using lectin histochemical techniques. In the horse, the duodenal gland was distributed in the area from the uppermost part of the small intestine to a point about 6m caudal to the pylorus. It consisted of two types of cells, mucous and serous cells. The former was found in glands distributed almost all over this part, but the latter was present in glands distributed restrictedly to the uppermost part of the small intestine at a point about 10 cm caudal to the pylorus. The cytoplasm of the mucous cell contained neutral glycoproteins with different saccharide residues as alpha-D-mannose, N-acetyl-beta (1----4)-D-glucosamine, galactose, alpha-galactose, alpha-N-acetylglucosamine, beta-D-Gal (1----3)-D-GalNAc, alpha-L-fucose and sialic acid. On the other hand, the serous cell contained neutral and acid glycoproteins with different residues such as alpha-D-mannose, alpha-D-glucose, beta-D-galactose(1----3)-D-N-acetylgalactose, alpha-L-fucose, N-acetyl-alpha-D-galactosamine, N-acetyl-beta (1----4)-D-glucosamine, galactose, alpha-galactose, alpha-N-acetylglucosamine and sialic acid. It is also elucidated in the present study that lipase, an enzyme for digestion, is contained in the serous cell of the equine duodenal gland.

A dense core vesicle protein is restricted to the cortex of granules in the exocrine atrial gland of Aplysia california.

We have generated a monoclonal antibody (mAb) 5E10 which recognizes an antigen localized to dense core vesicles (DCVs) in the atrial gland of Aplysia californica. mAb5E10 immunoprecipitates an abundant 57-kDa glycoprotein (atrial gland granule-specific antigen, AGSA) which is a soluble component of atrial gland DCVs. Electron microscopy reveals that AGSA immunoreactivity is restricted to the region between the dense core, which contains neuropeptide immunoreactivity, and the membrane of atrial gland DCVs. AGSA was purified by immunoaffinity chromatography, and the amino acid sequences of both N-terminal and internal cyanogen bromide fragments were determined. This information was used to isolate a 2.8-kilobase cDNA which encodes a 47-kDa protein. The predicted amino acid sequence contains the micro-sequenced peptides, an N-terminal hydrophobic signal sequence, and four N-linked glycosylation sites, but does not contain any significant homologies to database sequences. Northern blots and light level immunocytochemistry demonstrate that the AGSA gene is specifically expressed in the atrial gland. The identification of a protein localized to the cortex of DCVs suggests that this region has a specialized role in the function of these vesicles.

Isolation and structure of two novel 6-kDa dimeric peptides from the corpora cardiaca of the insect Locusta migratoria. Molecular mass determination by mass spectrometry.

We have isolated two major 6-kDa peptides from extracts of corpora cardiaca of adult females of Locusta migratoria. These peptides have been characterized by peptide sequencing and liquid secondary-ion mass spectrometry. They are structurally related dimers, one (6278.5 Da) being a homodimer (A-A chains), the other (6280.5 Da) being a heterodimer (A-B chains). A 60% similarity exists between the A and B chains. Both peptides have been chemically synthesized and the synthetic compounds appeared to be identical to the native ones. Polyclonal antibodies raised against each of these peptides demonstrated that they were contained within the secretory granules of the intrinsic cells of the glandular lobes of the corpora cardiaca. The physiological significance of these two peptides is unknown but, using the synthetic peptides, we are currently probing their biological role.

Complex formation with the fibroin gene enhancer through a protein-protein interaction analyzed by a modified DNA-binding assay.

The distal upstream region of the fibroin gene, -238 to -73, is known to contain the cis-acting element(s) responsible for tissue-specific transcription enhancement in vitro. To identify factor(s) trans-acting on this region, we have developed a modified nitrocellulose blot protein-DNA binding procedure that detects DNA-protein complexes via protein-protein interaction. A protein that does not bind to DNA as a monomer cannot be detected by standard Southwestern blotting. To detect protein-DNA complexes involving both protein-DNA and protein-protein interactions, the protein blot was reacted with the extract protein to form a protein complex on the filter, in the presence of the DNA probe. This new method revealed that the 75-kDa protein binds to the enhancer region of the fibroin gene in the presence of the second protein(s). The 75-kDa protein and the factor(s) mediating DNA binding activity were recovered in two different column fractions. The level of the 75-kDa protein appeared to be much higher in silk gland extracts than in nonsilk gland extracts, whereas the mediating factor(s) distributed evenly. These observations suggest that the distal upstream region of the fibroin gene can be specifically recognized by a protein complex composed of at least two distinct proteins, through protein-protein interaction.

Tetranectin immunoreactivity in normal human tissues. An immunohistochemical study of exocrine epithelia and mesenchyme.

A recently discovered human plasma protein, tetranectin (TN), which has previously been demonstrated immunohistochemically within various endocrine tissues, was in this study identified in an additional number of epithelial and mesenchymal cells by two polyclonal antibodies and one monoclonal using the conventional immunoperoxidase staining technique and a modification of the CLONO-GLAD procedure. TN was found in endothelial and epithelial tissues, particularly in cells with a high turn-over or storage function such as gastric parietal and zymogenic cells, absorptive surface epithelium of the small intestine, ducts of exocrine glands and pseudostratified respiratory epithelium. Also mesenchymal cells produced a TN positive staining reaction, which was most conspicuous in mast cells, but also present in some lymphocytes, plasma cells, macrophages, granulocytes, striated and smooth muscle cells and fibroblasts. SDS-PAGE and Western blotting analysis of cultured human embryonal fibroblasts (WI-38) showed that the cells besides TN contain another protein with a molecular weight of 82,000. As this protein, however, reacted with our affinity purified antibodies it probably represents a precursor of TN or a protein with a molecular weight of approximately 60,000, which is covalently linked to TN. This and the fact that TN shows amino acid sequence homologies to the carboxyterminal part of the asialo-glycoprotein receptor and a cartilage proteoglycan core protein as well a binding affinity to plasminogen points to TN as being part of a larger molecule, which possibly has been cleaved by proteolysis at the cellular site and then passed into the blood, where it polymerizes into a tetramer.

Single-step purification of two hyperglycaemic neurohormones from the sinus gland of Procambarus bouvieri. Comparative peptide mapping by means of high-performance liquid chromatography.

A crude aqueous extract from 2000 sinus glands of the Mexican crayfish Procambarus bouvieri (Ortmann) was fractionated on a muBondapak-Phenyl column. Two isoforms of the crustacean hyperglycaemic hormone, designated CHH-B and CHH-C in order of elution, were isolated in pure form. Their biochemical characterization showed a remarkable degree of homology. A tryptic digest of each isohormone was fractionated on an Ultrasphere-ODS column. Only one tryptic peptide in CHH-C was eluted later than its homologous peptide in CHH-B. On acid hydrolysis both tryptic peptides had the same composition but, as they contain Asp and Glu, we suspect that the difference residues in a double reciprocal amidation-deamidation of two acidic residues.

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.

Interaction of composite protein complex with the fibroin enhancer sequence.

To characterize proteins that bind to the upstream region of the fibroin gene, we used an electrophoretic mobility assay and a DNase I footprinting assay. A crude nuclear extract from the posterior silk glands forms a sequence-specific complex with the upstream region, -234 to -66, which includes a signal for tissue-specific transcriptional enhancement. This specific complex is composed of at least two factors of proteinous nature and the DNA. The composite proteins specifically protect an approximately 13-base pair (-167 to -155) region in a DNase I footprinting assay. This region contains an interesting repeated sequence, AATTTAATTT. Crude nuclear extracts from the middle silk glands and the ovarian tissues, but not a HeLa whole cell extract, also give the band complexes of similar electrophoretic mobility to the one formed in the posterior silk gland extract. However, under a higher EDTA condition, these complexes are more unstable than the one constructed in the posterior silk gland extract. These results suggest a possibility that these proteinous molecules in the complex, although apparently ubiquitous in Bombyx cells, might be specifically modified in the posterior silk gland cells to play an important role in the specific expression of the fibroin gene.

In vitro transcription of eukaryotic genes is affected differently by the degree of DNA supercoiling.

In a posterior silk gland extract, covalently closed circular (ccc) DNA is in a superhelical state that supports more transcription of fibroin gene than does linear DNA. A HeLa cell extract showed neither the supercoiling activity nor the preference for the transcription of ccc DNA over linear DNA. These activities could be added to the HeLa cell extract. Phosphocellulose fractionation of the posterior silk gland extract yielded a flow-through fraction and a 0.6 M KCl eluate fraction that were required for the supercoiling and for the efficient transcription of the ccc template in the acceptor HeLa cell extract. The 0.6 M KCl fraction had a DNA topoisomerase II activity, and the flow-through fraction contained a supercoiling factor that, with the aid of topoisomerase II, introduced negative supercoils into ccc DNA. When both fractions were added to the posterior silk gland extract, more supercoiling occurred than with the extract alone. Several genes were optimally transcribed under various extents of supercoiling. The fibroin gene and adenovirus 2 major late promoter were fully transcribed as partially supercoiled templates. The sericin gene required more supercoiling for full transcription, whereas no preference for supercoiling was seen with the transcription of hsp70. These results suggest that DNA topology plays a role in the regulation of gene expression.

Metasternal gland secretion of the locust tree borer, Megacyllene robiniae (Coleoptera: Cerambycidae).

1. The volatile components of metasternal gland extracts of male and female Megacyllene robiniae have been analyzed by gas chromatography-mass spectroscopy. 2. The major component was identified as 2-(1,3-hexadien-1-yl)-5-methyltetrahydrofuran, a new natural product. 3. 1-Phenylethanol is present only in male extracts. 4. Acetates of hexadecanol and octadecanol are also present.

Lectinhistochemical observations on the bronchial glands and the goblet cells of the airway epithelium of marine diving mammals.

Carbohydrate components were investigated in the bronchial glands and goblet cells of two species of seals with different diving habits (Weddell seal, Crabeater seal). Bouin-fixed tissue was incubated with various fluorescein-iso-thiocyanate-conjugated lectins (WGA, RCA I, PNA, PHA I, HPA, GSA I, DBA, Con A, LFA, SBA, UEA I), additionally PAS- and HID-reactions as well as Alcian Blue stains were applied. The two species differ in respect of their carbohydrate histochemistry, e.g., the PAS reaction gives only a faint stain at the apical membrane in the bronchial glands of the Weddell seal, whereas in the Crabeater seal several cells give a positive reaction in the entire cytoplasm; or RCA I has only few binding sites at the apical membranes of the bronchial glandular cells of the Weddell seal whereas many cells of Crabeater bronchial glands react strongly positive. It is concluded that the secretory products of the bronchial glands of the long- and deep-diving Weddell seal are relatively poor in mucins, which is in contrast to other mammals studied so far.

The preparation of poly(A)+mRNA from the hagfish slime gland.

The isolation of translatable poly(A)+mRNA from the slime glands of the Pacific hagfish, Eptatretus stouti, is not possible by the commonly used procedures because of the viscous slime that is formed when the contents of the glands are hydrated. This paper reports on a procedure developed to overcome this problem. Briefly, the tissue was powdered in liquid nitrogen, mixed with sodium lauroylsarcosine and proteinase K and lyophilized. The lyophilized powder was then mixed with 0.3 mm diameter glass beads, thoroughly ground and wetted with buffer and digested at 37 degrees C. The RNA from the digest was recovered by ultracentrifugation through a CsCl cushion. Further purification of the RNA was accomplished by the usual methods with slight modifications.

Maturation of hagfish gland thread cells: composition and characterization of intermediate filament polypeptides.

Previous studies with the hagfish, a primitive vertebrate, have shown that the gland thread cells (GTCs) each contain a single thread (approximately 60 cm long in average-sized cells) in the form of a concisely coiled cytoskeletal entity destined for export by holocrine secretion. The thread in relatively immature GTCs consists almost entirely of intermediate filaments (IFs) bundled in parallel alignment with far fewer microtubules (MTs). The three thread polypeptides described earlier (alpha, basic; beta, acidic; gamma, most acidic; each with a Mr of 63-64 kD) are now further evaluated with respect to in vitro assembly, cross-reactivity with IF polypeptides from higher vertebrates, and peptide sequence homology with known IF polypeptides. The overall results mainly suggest that the hagfish polypeptides are keratinlike substances but lamins or a new type of IF is not ruled out. However, cross-reactivity is weak with mammalian keratins; the 8-11-nm filaments formed from mixtures of alpha and gamma in vitro are generally linear rather than the curvilinear structures usually formed by keratin and nonkeratin IFs; and mixtures of alpha and beta tend to yield 9-12-nm granules or granular strings. Polypeptide analyses on GTCs segregated on the basis of maturational stage show a progressive increase in beta/gamma values which correlates with cell maturation, but the alpha/(beta + gamma) ratios remain near 1. Inasmuch as beta and gamma have many similar properties, the documented increase in the amount of the beta component in aging GTCs might in part be the result of a failure in a posttranslational modification system and may contribute to the ultrastructural changes that accompany thread maturation in preparation for holocrine secretion and subsequent modulation of the viscoelastic properties of mucus.

Purification and the partial amino acid sequence of an insect neurotoxin from the venom of scorpion Buthus martensi Karsch.

1. A neurotoxic peptide was isolated from the venom of the scorpion Buthus martensi Karsch collected in Henan Province, China. 2. This toxin showed the highest neurotoxic potency to crickets amongst all components in the venom examined. 3. The amino acid composition of the toxin was similar to that of insect toxin 1 of Leiurus quinquestriatus quinquestriatus. 4. The partial primary sequence of the toxin at the N-terminal was very similar to that of an insect toxin of Androctonus australis Hector. 5. We conclude that the neurotoxin we isolated is indeed an insect toxin and thus named it as BmK IT.

Morphological examination of intrahepatic bile ducts in hepatolithiasis.

Numerous glandular elements are characteristically found within and around the intrahepatic bile duct walls in hepatolithiasis. These glandular elements were studied by reconstruction of serial sections and mucus histochemistry. The glands were of two types: glands within the thickened ductal wall (intramural) and those outside the wall (extramural). The former were mucous glands arranged in tubular pattern and the latter seromucous glands arranged in tubuloalveolar pattern. Mucous acini of both glands were rich in neutral, carboxylated and sulfated mucus glycoproteins. Serial section observations showed that the intramural glands communicated with bile duct lumina directly, and the extramural glands with ductal lumina via their own conduits. The intramural glands were usually continuous with the epithelia lining bile ducts, suggesting that they were derived from an invagination and subsequently proliferating epithelium lining bile ducts. The extramural glands may have arisen from a proliferation of the pre-existing peribiliary glands. Hypersecreted mucus from the intramural and extramural glands might be causally related to the development and growth of calculi in the intrahepatic biliary tree.