Bruce Bogart

Secretory dynamics of the rat submandibular gland. An ultrastructural and cytochemical study of the isoproterenol-induced secretory cycle

Upon isoproterenol induction of secretion, there was an explosive exocytosis. The lumen greatly expanded during this stage, penetrating deeply into the cell cytoplasm. The intermediate stage was characterized by a reduction in the size of the lumen, an increase in the Golgi apparatus, and the presence of small vesicles and endocytotic profiles in close association with the lumen. An increase in lysosomes and GERL was verified by their acid phosphatase activity. The final stage consisted of repopulation of secretory granules. Overlying the cyclical distribution of secretory granules was the cyclical changes in the morphology of the lateral intercellular spaces, the localization of cation visualized by pyroantimonate precipitation, and the localization of K+-dependent oubain-sensitive p-nitrophenylphosphatase. The implication of a lysosomal function in processing retrieved membrane after exocytosis and the possibility that the lateral intercellular spaces were functioning as backward channels during fluid transport were discussed.

The Biologic Activities of Cystic Fibrosis Serum. I. The Effects of Cystic Fibrosis Sera and Calcium Ionophore A 23187 on Rabbit Tracheal Explants

Summary: An ionophore A23187-induced increase in membrane permeability to calcium ions in culture medium produced a rabbit tracheal mucociliary response indistinguishable from that caused by cystic fibrosis (CF) sera on three different occasions. Specific chelation of calcium ions with EGTA in the basal medium Eagle (BME) media with no additive or in native CF sera abolished the mucociliary disturbances in all cases. Increased membrane permeability to calcium may be important in the production of the mucociliary response by CF serum factor(s) in the tracheal assay system.Speculation: CF serum factor(s) may be acting on the cultured rabbit tracheal explant cellular membranes to produce altered permeability to ions. This alteration in membrane permeability may be promoting a loss of intracellular communication and cellular injury. Such changes on the cellular level may be related to the pathophysiology of this genetic disorder.

Changes in the diurnal pattern of the distribution of gelatinases and associated proteins in normal and pathological tear fluids: evidence that the PMN cell is a major source of MMP activity in tear fluid.

The matrix metalloproteases (MMP) play critical roles in modulating apoptosis, angiogenesis, cell migration, wound healing, tissue remodeling and inflammation in all connective tissues. Two extracellular MMPs, both type IV gelatinases, MMP-2 and MMP-9 (gelatinase A and B), as well as their respective inhibitors TIMP-1, TIMP-2 and TIMP-3 have been detected in the human cornea. MMP-9 is localized in the epithelium while MMP-2 is expressed preferentially by stromal keratocytes, and to a lesser extent, in the epithelium with expression of both enzymes up-regulated during wound healing.1 MMPs, principally pro-MMP-9 and associated complexes, have been detected in human tear fluid in the open eye condition. Active MMP-9 is presumed to be derived principally from the epithelium. Increased levels of enzyme and a shift in the pattern of distribution from proenzyme to the active MMP-9 species, along with the emergence of pro-MMP-2 and active MMP-2, has been observed in tear fluid in a wide range of pathological conditions. These conditions include external ocular infections, corneal wound healing secondary to LASIK, sterile corneal ulcers, dry eye syndrome, active keratoconus, ocular rosacea and sterile corneal melts.2–4 This study was designed to examine the origins and the nature of gelatinolytic species and associated proteins present in normal and pathological tear fluid during open and closed eye phases of the diurnal cycle.

Interaction and release of soluble immune complexes from mouse B lymphocytes

Abstract Soluble immune complexes ( 125 I BSA-anti-BSA-C) bind to B lymphocytes and accumulate at one pole of the cells (“caps”). The complexes remain on the membrane after incubation of the cells at 37 °C in tissue culture medium for several hours. The 125 I BSA can be quantitatively removed from the cell surface by incubation with excess BSA but not with excess antibody to BSA or preformed BSA-anti-BSA-C complexes. The release of 125 I BSA is probably due to the removal of the complexes from the cell membrane and not to an exchange between unlabeled BSA in the medium and the labeled BSA present in the membrane-bound complexes. Release of 125 I BSA by excess BSA is temperature dependent. The membrane-bound complexes can also be removed by incubating the cells with papain fragments of rabbit antibody to mouse Ig (anti- γ 1 , γ 2 , and k Ig chains). However, after exposure to divalent [F(ab′) 2 or 7S Ig] rabbit antibodies to mouse Ig, the complexes remain associated with the cells. In addition, after such treatment the complexes cannot be removed by excess BSA or by Fab anti-Ig.

Biological Activities of Cystic Fibrosis Serum. IV. Stimulation of the Calcium Mediated K + Efflux from Rat Submandibular Gland Fragments

Summary: Cystic fibrosis (CF) and heterozygote sera stimulate a significant K+ efflux from rat submandibular gland fragments in the presence of 1 mM ouabain. This sensitive parameter can be maximally stimulated by as little as 0.5% CF serum and is inhibited by the calcium channel blocker D600 and EGTA. Specific receptor blockers propranolol, phenoxybenzamine or atropine do not inhibit the CF serum-stimulated K+ efflux and agonists do not supramaximally stimulate K+ efflux when added with serum. CF serum-induced K+ efflux did not result in the leakage of lactic dehydrogenase into the bathing media nor did it mimic the action of the calcium ionophore A23187 when added in the presence of D600. In addition, ultrafiltrates of CF serum (less than 10,000 daltons) also stimulated K+ efflux from rat submandibular gland tissue fragments.Speculation: Cystic fibrosis serum factor(s) may be altering membrane permeability to calcium which results in the release of K+ from rat submandibular gland fragments.

The Biologic Activities of Cystic Fibrosis Serum. II. Ultrastructural Aspects of the Effect of Cystic Fibrosis Sera and Calcium Ionophore A23187 on Rabbit Tracheal Explants

Summary: Ultrastructural and cytochemical observations indicate that both cystic fibrosis (CF) sera and calcium ionophore A23187 induce a swelling or an increase in the size and possibly the number of secondary lysosomes and an increase in mucus secretion in epithelium of the rabbit tracheal bioassay system. Extended incubation of the rabbit tracheal explants with either CF or control sera produces a cytotoxic effect on the tracheal epithelium, but only after the termination of the normal bioassay time period. Comparative ultrastructural study of the effect of both CF sera and calcium ionophore A23187 on the rabbit tracheal bioassay system indicates that increased membrane permeability to calcium may be important in the production of the ciliary dyskinesia response by CF serum factor(s) in the rabbit tracheal bioassay system.Speculation: CF serum factor(s) may be acting on the cultured rabbit tracheal explant epithelial membranes to produce altered membrane permeability to ions and to calcium in particular. This alteration in membrane function may be promoting the observed ultrastructural changes in the epithelial cells which include a swelling or increase in the number and size of secondary lysosomes and an increase in mucus secretion as well as ciliary dyskinesia. Alterations in membrane function and permeability may be related to the pathophysiology of this genetic disorder.

Is the cystatin-like domain of TSL functionally active in external ocular infections and during the normal diurnal cycle?

PURPOSE To test whether the cystatin-like functional domain in tear specific lipocalin (TSL) is functionally active in tears during the normal diurnal cycle and during external ocular infections. METHODS Capillary tube collected reflex (RTF), open (OTF) and closed (CTF) eye tear samples were recovered from six normals and semi-quantitatively western blot assayed for cystatin C and TSL. CTF samples were immunoprecipitated with antibodies raised against TSL, cystatin C and other antiproteases and screened for the co-precipitation of proteases by casein and gelatin zymography. OTF samples recovered from individuals with viral, fungal and bacterial keratitis were similarly screened for TSL-bound proteases. Human tissue was subjected to immunohistochemical study. RESULTS Western blot analysis reveals a progressive increase in cystatin C in going from RTF to OTF to CTF samples (approximately 3, 7 and 30 ng microl(-1), respectively). In contrast, the concentration of TSL remains constant (approximately 1500 ng microl(-1)). Immunocytochemistry data show staining of the apical surface of the human conjunctiva and some intra-cellular staining for cystatin C, but not for cystatin A. Zymography confirms earlier data that CTF contains exceptionally high levels of proteases bound to a wide range of specific inhibitors. However, only trace amounts of proteases are complexed with cystatin C and no protease can be detected bound to TSL in either the pathological or CTF samples. CONCLUSION Although TSL contains a functional cystatin-like domain, it is not physiologically active during the normal diurnal cycle or during external ocular infections. Reactive proteases in CTF are most likely controlled by the presence of excess levels of more reactive cystatins, especially cystatin C, which accumulates during prolonged eye closure. Immunohistochemical data suggest that the apical conjunctiva may be a contributing source for the accumulating cystatin C.

Biological activities of cystic fibrosis serum. III. CF serum induced uptake of 45Ca++ by rabbit tracheal explants.

Abstract Cystic Fibrosis (CF) serum and its isolated component IgG fraction produce an increased uptake of 45 Ca ++ in rabbit tracheal explants when compared to control serum and its isolated IgG fraction. Heterozygote serum also produced an increased uptake of 45 Ca ++ but not to the same extent as CF serum. The calcium channel blocker D600 inhibited the CF serum induced uptake of 45 Ca ++ indicating that CF serum may be acting on the plasma membrane to produce changes in calcium permeability in rabbit tracheal explants.

Characterization and origin of major high-molecular-weight tear sialoglycoproteins.

The purpose of this study was to characterize the nature and origin of the major high molecular weight soluble sialoglycoproteins in the tear fluid in open and closed eye environments.

High-performance liquid chromatographic fractionation and partial characterization of cystic fibrosis serum ultrafiltrates

Analytical separation of serum ultrafiltrates by high-performance liquid chromatography produces a distinctive peak with a retention time of 18.5-21 min (subfraction 18.5) from cystic fibrosis serum ultrafiltrates and obligate heterozygote serum ultrafiltrates, but not in significant concentrations from control or asthmatic serum ultrafiltrates. Semipreparative separation of control serum ultrafiltrates produced a small peak with similar retention time that was approximately 1% of the arbitrary absorbance units found in this cystic fibrosis subfraction. Subfraction 18.5 had biological activity only when separated from cystic fibrosis serum ultrafiltrate, but did not contain measurable amounts of C3a des-arginine and C4a des-arginine. Subfraction 18.5 is a low-molecular-weight material (1000-1400 daltons) that contains 14.9 micrograms orcinol positive material per 50 micrograms protein. The spectrum of subfraction 18.5 indicates that it has to be purified to homogeneity.