Biogenesis of the protein storage vacuole crystalloid.

We identify new organelles associated with the vacuolar system in plant cells. These organelles are defined biochemically by their internal content of three integral membrane proteins: a chimeric reporter protein that moves there directly from the ER; a specific tonoplast intrinsic protein; and a novel receptor-like RING-H2 protein that traffics through the Golgi apparatus. Highly conserved homologues of the latter are expressed in animal cells. In a developmentally regulated manner, the organelles are taken up into vacuoles where, in seed protein storage vacuoles, they form a membrane-containing crystalloid. The uptake and preservation of the contents of these organelles in vacuoles represents a unique mechanism for compartmentalization of protein and lipid for storage.

The protein storage vacuole: a unique compound organelle.

Storage proteins are deposited into protein storage vacuoles (PSVs) during plant seed development and maturation and stably accumulate to high levels; subsequently, during germination the storage proteins are rapidly degraded to provide nutrients for use by the embryo. Here, we show that a PSV has within it a membrane-bound compartment containing crystals of phytic acid and proteins that are characteristic of a lytic vacuole. This compound organization, a vacuole within a vacuole whereby storage functions are separated from lytic functions, has not been described previously for organelles within the secretory pathway of eukaryotic cells. The partitioning of storage and lytic functions within the same vacuole may reflect the need to keep the functions separate during seed development and maturation and yet provide a ready source of digestive enzymes to initiate degradative processes early in germination.

Identification of R-GRAMP, a membrane glycoprotein of regulated secretory granules in primate cells.

Investigation of membrane assembly and traffic in the regulated secretory pathway may be facilitated by identification of membrane components that are unique to regulated secretory granules. To identify such markers, we isolated integral membrane proteins by Triton X-114 extraction from well-differentiated monolayers of an exocrine cell line, the goblet cell subclone (18N2) of the human colon carcinoma cell line HT29, and used the extracts as immunogens to produce monoclonal antibodies (mAbs). Immunofluorescence microscopy of HT29 goblet cell monolayers identified one mAb (MG-1) that labeled a component of mucin granule membranes. Immunofluorescence of frozen semithin sections of normal intestine, and various other human and monkey tissues, showed that this antigen is present in regulated secretory granule membranes of primate exocrine cells, endocrine cells, and tissue granulocytes. EM immunogold labeling of goblet cells, enteroendocrine cells and eosinophils confirmed that the antigen is associated with secretory granule membranes and not with plasma membranes. The antigen was identified by SDS-PAGE autoradiography of immunoprecipitates from HT29 goblet cells metabolically labeled with [35S]methionine and [35S]cysteine or [3H]glucosamine, as a glycoprotein with an apparent molecular mass ranging from 23 to 37 kDa. Digestion of immunoprecipitates with N-glycosidase F reduced the apparent mass to 16 to 19 kDa. This small, highly-glycosylated protein was named "R-GRAMP" (for regulated granule-associated membrane protein) to reflect its wide distribution in secretory granule membranes of regulated exocrine, endocrine and granulocytic cell types. This distribution suggests that it may play a common functional role in regulated secretion.

Effect of cyclosporine on conjunctival mucin in a canine keratoconjunctivitis sicca model.

PURPOSE: To test the hypothesis that beneficial effects of Cyclosporin A (CsA; Sandimmune; Sandoz, Basel, Switzerland) in treating keratoconjunctivitis sicca (KCS) include an effect on the mucin-producing conjunctival goblet cells independent of CsA's effect on lacrimation. METHODS: Keratoconjunctivitis sicca was induced bilaterally in six dogs after removal of orbital and nictitans lacrimal glands. Two weeks after induction of KCS, either 2% CsA or vehicle was applied twice daily to each surgically altered eye until 6 weeks after KCS induction. Eyes of three control dogs without surgically altered eyes were treated twice daily with vehicle only. Incisional biopsy specimens of ventral fornix conjunctiva were collected before gland removal (baseline) and at 2, 4, and 6 weeks after KCS induction. At each sampling time, eyes were photographed, and color images were subsequently graded for degree of conjunctivitis and characteristics of ocular discharge. Intracellular mucin stores in conjunctival epithelia were estimated using computer-assisted morphometry of biopsy specimen cross sections, and clinical and morphometric findings were correlated. RESULTS: Lacrimal gland removal resulted in induction of KCS in dogs by 2 weeks, with mean Schirmer tear test (STT) values of 5 mm/min or less occurring in surgically altered eyes compared with STT values of 22.5 mm/min before surgery and 22.9 mm/min in unaltered control eyes at 2 weeks. In surgically altered eyes, STTs remained low during the 6-week study, independent of topical treatment. Intracellular mucin stores were quantified from conjunctival samples collected from each eye at baseline and 2, 4, and 6 weeks. At 4 and 6 weeks (after 2 and 4 weeks of topical treatment), intraepithelial mucin quantities were significantly greater (P: < 0.05) in CsA-treated KCS eyes (14.4 and 13.1 microm(2)/microm, respectively) compared with pretreatment KCS (7.4 microm(2)/microm) eyes and vehicle-treated KCS eyes (7.3 and 8.5 microm(2)/microm, respectively). KCS eyes treated with CsA had lower conjunctivitis and ocular discharge scores than did vehicle-treated KCS eyes. CONCLUSIONS: Topical 2% CsA restored in vivo conjunctival mucin stores to control levels over a 4-week period, determined by computer-assisted morphometry of sequential conjunctival biopsy specimens from eyes of dogs with surgically induced KCS. Degree of conjunctivitis and severity of mucus discharge were decreased in KCS eyes treated with CsA. Because lacrimal tissues were removed from animals in this study, conjunctival responses occurred independent of lacrimogenic effect(s). These results indicate that restoration of conjunctival goblet cell mucin production, i.e., the balance between synthesis and secretion of mucin glycoproteins, may play an important role in the beneficial effect of CsA in treating KCS.

On the conductance pathway traversed by strontium in mediating the asynchronous release of acetylcholine by motor nerve impulses.

A study was made to determine whether the Sr2+ dependent asynchronous release of acetylcholine by nerve impulses is mediated by the conventional Ca2+ conductance channel or, as has been suggested recently, through an alternative ion pathway. Experiments were performed on the frog neuromuscular junction by the use of standard electrophysiological techniques. Repetitive nerve stimulation in Sr2+-Ringer solutions caused a marked increase in miniature end-plate potential (m.e.p.p.) frequency which was dependent on Sr2+ concentration and inhibited in a competitive fashion by the known Ca2+ antagonists, Co2+ and Mg2+. The equilibrium dissociation constants (KdS) determined for both Co2+ (0.09 +/- 0.01 mM, mean +/- s.e. mean, n = 5) and Mg2+ (3.7 +/- 0.3 mM, mean +/- s.e. mean, n = 4) were essentially the same as the reported values for these antagonists in blocking Ca2+ -mediated transmitter release by nerve impulses. These results suggest that Sr2+ mediates asynchronous evoked transmitter release through the conventional calcium conductance channel.

The effect of 16,16-dimethyl prostaglandin E2 on proliferation of an intestinal goblet cell line and its synthesis and secretion of mucin glycoproteins.

The effect of 16,16'-dimethyl prostaglandin E2 (dmPGE2) on the human colonic adenocarcinoma derived mucus-secreting goblet cell line HT29-18N2 was investigated. The proliferation rate of HT29-18N2 was increased by exposure to 10 or 100 microM dmPGE2. Exposure to 10 or 100 microM dmPGE2 caused a significant decrease in the rate of radiolabeled glucosamine incorporation into newly synthesized glycoproteins during an 8 or 24 h exposure. At concentrations as low as 1 microM, dmPGE2 accelerated the secretion of mucin glycoproteins as assessed by the release of newly synthesized radiolabeled glycoproteins, a mucin-specific enzyme-linked immunoassay and a whole-mount immunofluorescence assay. A 1 h exposure to dmPGE2 did not, however, result in a morphometrically detectable decrease in intracellular mucous granule stores or elicit any other readily detectable morphological change. The experimental results suggest elevated levels of PGs may contribute to the previously recognized decreases in intracellular mucin stores and shifts in the types of mucins species present at sites of mucosal inflammation in ulcerative colitis patients.

Lipoxygenase metabolites of arachidonic acid do not induce mucus secretion from rabbit intestinal goblet cells in vitro.

Lipoxygenase metabolites of arachidonic acid are effective mucus secretagogues in the respiratory tract but their efficacy in the intestinal tract was unknown. Mucosal explants and sheets of epithelial cells isolated from rabbit small and large intestine were exposed to leukotrienes B4, C4, and D4 and monohydroxyeicosatetraenoic acids 5-HETE, 12-HETE, and 15-HETE. Light and electron microscopic inspection of goblet cells in treated tissues failed to detect evidence of recent compound exocytosis of mucin granules or other morphological evidence of secretory activity. These results indicate that lipoxygenase metabolites are not directly responsible for the increased mucus secretion observed in ulcerative colitis.

An automated, handheld biosensor for aflatoxin.

A new immunoaffinity fluorometric biosensor has been developed for detecting and quantifying aflatoxins, a family of potent fungi-produced carcinogens that are commonly found in a variety of agriculture products. They have also been cited as a biological agent under weapons development. The handheld, self-contained biosensor is fully automatic, highly sensitive, quick, quantitative, and requires no special storage. Approximately 100 measurements can be made before refurbishment is required, and concentrations from 0.1 parts per billion (ppb) to 50 ppb can be determined in <2 min with a 1 ml sample volume. The device operates on the principles of immunoaffinity for specificity and fluorescence for a quantitative assay. The analytic procedure is flexible so that other chemical and biological analytes could be detected with minor modifications to the current device. Advances in electro-optical components, electronics, and miniaturized fluidics were combined to produce this reliable, small, and versatile instrument.

Success of preoperative imaging and unilateral neck exploration for primary hyperparathyroidism.

The surgical treatment of hyperparathyroidism has become controversial with the recent advent of reliable preoperative imaging modalities. This study examines the efficacy and economy of using preoperative imaging studies to localize the pathology and allow for unilateral neck exploration. From January 1990 to May 1996, a total of 91 patients with primary hyperparathyroidism were treated at Swedish Medical Center in Seattle, WA, by 2 surgeons. Eighty-six nuclear scintigraphy studies were performed, of which 44 were technetium 99m sestamibi (Tc-99m-sestamibi) scans and 42 were thallium 99m technetium (Th-99m-Tc) scans. The overall sensitivity for Tc-99m-sestamibi was 91% (40/44), and that for Th-99m-Tc scans was 81% (34/42). Ultrasound examination revealed a sensitivity of 80% (66/82). There was a statistically significant difference in surgical time between the unilateral and bilateral neck explorations (45 minutes, P < 0.0001). Unilateral neck exploration for hyperparathyroidism has been successful in curing hypercalcemia 93% (85/91) of the time with the use of preoperative imaging studies. Tc-99m-sestamibi is a reliable tool for planning the initial unilateral neck exploration for treatment of primary hyperparathyroidism.

Versatile four-probe ac conductivity measurement system.

The design of a four-probe ac conductivity measurement system is described. The system is capable of making precision conductance measurements from 10(-8) to 10(-5) S at 20 to 200 Hz. Driven coaxial shields and a controlled current source are utilized to eliminate phase shift/attenuation errors, and make the measurement of conductances on a variety of materials a routine procedure requiring a minimum of operator intervention.

Effect of intermittent fasting on prostate cancer tumor growth in a mouse model.

Caloric restriction (CR) has been shown to have anti-cancer properties. However, CR may be difficult to apply in humans secondary to compliance and potentially deleterious effects. An alternative is intermittent CR, or in the extreme case intermittent fasting (IF). In a previous small pilot study, we found 2 days per week of IF with ad libitum feeding on the other days resulted in trends toward prolonged survival of mice bearing prostate cancer xenografts. We sought to confirm these findings in a larger study. A total of 100 (7- to 8-week-old) male severe combined immunodeficiency mice were injected subcutaneously with 1 × 10(5) LAPC-4 prostate cancer cells. Mice were randomized to either ad libitum Western Diet (44% carbohydrates, 40% fat and 16% protein) or ad libitum Western Diet with twice-weekly 24 h fasts (IF). Tumor volumes and mouse bodyweights were measured twice weekly. Mice were killed when tumor volumes reached 1000 mm(3). Serum and tumor were collected for analysis of the insulin/insulin-like growth factor 1 (IGF-1) hormonal axis. Overall, there was no difference in mouse survival (P=0.37) or tumor volumes (P ≥ 0.10) between groups. Mouse body weights were similar between arms (P=0.84). IF mice had significantly higher serum IGF-1 levels and IGF-1/IGFBP-3 ratios at killing (P<0.001). However, no difference was observed in serum insulin, IGFBP-3 or tumor phospho-Akt levels (P ≥ 0.39). IF did not improve mouse survival nor did it delay prostate tumor growth. This may be secondary to metabolic adaptations to the 24 h fasting periods. Future studies are required to optimize CR for application in humans.

Both crypt and villus intestinal goblet cells secrete mucin in response to cholinergic stimulation.

Computer-assisted morphometric analysis was used to quantify the effects of cholinergic stimulation on intestinal goblet cells. Within 5 min of stimulation (250 micrograms/kg carbachol sc), many crypt goblet cells were depleted of mucin secretory granules and their apical membranes had the deep cavitation that accompanies recent compound exocytotic activity. The percentage of crypt epithelial volume occupied by mucin secretory granules was decreased by 58.4% at 5 min and 45.9% at 60 min. Although villus goblet cells never showed signs of recent compound exocytosis, morphometric analysis revealed a 22.4% decrease in the percentage of villus epithelial volume occupied by mucin secretory granules within 5 min of stimulation and a 32.4% decrease by 60 min. The decrease in villus mucin stores was due to both a reduction in the volume of mucin in an average villus goblet cell and a drop in the number of recognizable goblet cells per square micrometer of villus epithelium. Mucin stores in both the crypt and villus regions were largely replenished by 4 h poststimulation.

Lectin binding patterns to plasmalemmal glycoconjugates of goblet cells undergoing differentiation in vitro.

The plasmalemmal glycoconjugates of the HT29-18N2 (N2) cell line were characterized on cells grown as 1) undifferentiated multilayers in glucose-containing culture media and 2) monolayers of columnar cells acquiring the goblet cell phenotype in glucose-free media. Lectins were unable to bind sheets of detached N2 cells in the absence of fixation. Following fixation with aldehydes, a dramatic unmasking of lectin binding sites was seen. When fixed monolayers were stained prior to embedding, biotinylated lectins, visualized by the avidin-biotin-complexed peroxidase technique, were more efficient than collodial gold-coupled lectins. Lectin binding sites could also be detected by using collodial gold-coupled lectins to stain monolayers embedded in LR White, Lowicryl K4M, and Lowicryl HM20. The binding of 5 lectins (wheat germ, Dolichos bifluros, peanut, soybean, and Ulex europeus) was found to be independent of the stage of differentiation; "pre-differentiated" columnar cells which had prominent microvilli and no or few mucous secretory granules had identical staining patterns as well-differentiated goblet cells with large numbers of secretory granules. Ricinus communis I was the only lectin whose binding was influenced by the stage of differentiation; it intensely labeled undifferentiated multilayers of N2 cells but only weakly labeled basolateral membranes of differentiated monolayers. Canavalia ensiformas (ConA) caused a moderate and even labeling of both apical and basolateral membranes of fixed monolayers stained prior to embedding, but post-embedding labeling revealed heavy labeling along the lateral margins of all columnar cells and weak to moderate binding along the apical and basal cell surface.

Morphometric analysis of mucous granule depletion and replenishment in rat colon.

Computer-assisted morphometric analysis was used to quantify mucous granule discharge and the subsequent replenishment of secretory granules in the rat colon following cholinergic challenge. Within 5 min of stimulation (250 micrograms/kg carbachol, subcutaneous), the volume of intracellular mucous granules decreased to 61.5% of the control. The apical plasma membranes of goblet cells in the midcrypt region became deeply cavitated, indicating that they had accelerated mucous granule secretion by a process of compound exocytosis. Goblet cells at the base of the crypt rarely showed cavitated apical membranes but clearly became depleted of intracellular mucous granules. At 60 min after stimulation, cavitated profiles were very rare (< or = 0.4%) but intracellular stores of mucous granules were still significantly depressed (54.7% of control). By 4 hr after stimulation, mucous granule stores recovered to 94.9% of control levels. Morphometric quantification was found to be a reliable and sensitive measure of recent goblet cell secretory activity.

Exocytosis and nerve terminal pseudopodia.

When exocytosis of synaptic vesicles is accompanied by the accumulation of vesicle membrane in the nerve terminal membrane, the geometric shape of the terminal must alter. The details of these rearrangements vary with the anatomical site; this laboratory has reported on the responses of abutted nerve terminals in the electric ray electric organ. When they are stimulated so as to lose synaptic vesicles, they develop reciprocal pseudopodial indentations (PSIs) with each other. Assuming that direct abutment of the interacting nerve terminals was necessary for this to occur, we have examined various nuclei of the rat brain limbic system for similar configurations. PSIs are most abundant between abutted terminals synapsing with smooth dendrites in the globus pallidus and substantia nigra. In these locations, there is good reason to believe that they are forming between swellings of the gamma-aminobutyric acid (GABA) afferents from the caudate-putamen. Conservative calculations of the potential accumulation of extracellular K released by action potentials at the PSI tip suggest that 15 mM concentrations could occur at firing rates of 150 Hz. Inasmuch as the GABA projection system to these nuclei is a system of boutons en passant, in which the safety factor for action potential conduction is low, it is suggested that the formation of PSI and the frequency-dependent accumulation of K could lower the safety factor to the point of action potential block. This may affect the inhibitory tone in the substantia nigra. An understanding of how PSI generation is regulated depends in part on knowing what options are available for synaptic vesicle behavior at the moment of depolarization of a nerve terminal. In particular, we need to know whether vesicles can open and close in situ during slow firing under physiological conditions. Recent experimental results enable us to foresee how this could be tested, and the experimental design is described.

Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line. Analysis using double labeling with lectins.

Lectins were used to characterize mucin glycoproteins and other secretory glycoconjugates synthesized by a human colon adenocarcinoma-derived cell line which expresses a goblet cell phenotype. Despite being clonally derived, HT29-18N2 (N2) cells, like normal goblet cells in situ were heterogeneous in their glycosylation of mucin. Only wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, and succinylated wheatgerm agglutinin, which binds N-acetylglucosamine, stained the contents of all secretory granules in all N2 goblet cells. The N-acetylgalactosamine binding lectins Dolichos biflorus and Glycine max stained 20% and 21% of N2 goblet cells respectively. Ricinus communis I, a galactose-binding lectin, stained 67% of N2 goblet cells although staining by another galactose-binding lectin, Bandeiraea simplicifolia I, was limited to 19%. Peanut agglutinin, a lectin whose Gal(beta 1-3)GalNAc binding site is not present on mucins produced in the normal colon but which is found on most mucins of cancerous colonic epithelia, stained 68% of the cells. Ulex europeus I, a fucose-binding lectin, did not stain any N2 goblet cells. Four lectins (Lens culinaris, Pisum sativum, Phaseolus vulgaris E, Phaseolus vulgaris L) which recognize sugars normally present only in N-linked oligosaccharides stained up to 38% of N2 goblet cells. The binding of these lectins indicates either both O-linked and N-linked oligosaccharide chains are present on the mucin protein backbone or the co-existence of non-mucin N-linked glycoproteins and O-linked mucins within the goblet cell secretory granule.

Selective secretion and replenishment of discrete mucin glycoforms from intestinal goblet cells.

Antibodies against MUC2, MUC3, and MUC5AC peptide epitopes stained the secretory contents of all goblet cells in the human colon-derived HT29-18N2 cell line. In contrast, four carbohydrate-specific monoclonal antibodies stained mucin glycoforms in consistent subsets of goblet cells. Cholinergic agonist-evoked decreases in total mucin stores were not always mirrored by proportional changes in mucin glycoforms in the same monolayers. Selective secretion of mucin glycoforms did not result from differences in receptor distribution, since cholinergic stimulation was found to increase intracellular free calcium in all cells and selective secretion was also observed when the cells were directly stimulated with the protein kinase C activator phorbol myristate acetate. The results demonstrate that goblet cells cycle through transient periods in which their exocytotic response is unresponsive to cholinergic or protein kinase C-mediated stimuli. Goblet cells replenished intracellular mucin stores to control levels within 1 h, but the relative proportion of mucin glycoforms was not always restored until 24 h after stimulation.

Signal transduction pathways mediating mucin secretion from intestinal goblet cells.

Cholinergic stimulation of the HT29-18N2 goblet cell line increased mucin secretion as assessed: (1) with a mucin-specific immunoassay, (2) using whole-mount immunocytochemistry, or (3) by morphometric quantification of intracellular mucous granule stores. Cholinergic stimulation did not, however, result in the apical plasmalemmal membrane cavitation that is characteristic of recent compound exocytotic activity. The response was not dependent on protein kinase C activation since it was not inhibited by the kinase C antagonist H7 or potentiated by the diacylglycerol kinase antagonist R59022. Calcium ionophore A23187 also accelerated mucin secretion by a noncompound exocytotic pathway. Activation of protein kinase C by phorbol 12-myristate 13-acetate, on the other hand, increased mucin secretion by a compound exocytotic pathway. The results provide insight into the signal transduction pathways underlying secretory responses of goblet cells observed in situ.

Bradykinin modulates mucin secretion but not synthesis from an intestinal goblet cell line.

The effect of the inflammatory mediator bradykinin on glycoprotein synthesis and mucin secretion in the human colonic adenocarcinoma cell line HT29-18N2 was examined. Bradykinin, at a threshold of 0.01 microM, accelerated the rate of mucin discharge as assessed by a mucin-specific ELISA. Using immunofluorescence microscopy, a thick meshwork of extracellular mucus was observed over bradykinin-treated monolayers but not mock-treated controls. Morphometric analysis of bradykinin-treated monolayers revealed no decreases in intracellular mucin stores or any other easily discernable morphological alteration. The ability of the cyclooxygenase inhibitors indomethacin and naproxen to decrease the response to bradykinin by approximately 68% indicates the effect is mediated, at least partially, through the generation of prostaglandins. Bradykinin did not alter the rate of incorporation of 3H-glucosamine into newly synthesized glycoproteins. Bradykinin-accelerated mucin secretion may be linked to the depletion of intracellular mucin stores in the inflammatory bowel disease ulcerative colitis.