Gene expression of coffee seed oxidation and germination processes during drying.

Coffee (Coffea arabica L.) seeds are sensitive to desiccation and oxidative stress during drying processes. We investigated the effect of drying and moisture levels on germination-related gene expressions associated with enzymatic systems that prevent oxidative stress in coffee seeds. Coffee seeds collected at physiological maturity were subjected to slow and quick drying to 40, 30, 20, and 12% moisture levels (wet basis), and as the control, seeds without drying were used. The seeds' physiological quality was calculated as percentage of normal seedlings at 15 and 30 days, normal vigorous seedlings at 30 days, and cotyledonary leaves at 45 days. The isoenzymes esterase, catalase (CAT), peroxidase (POX), and endo-ß-mannanase expressions were electrophoretically analyzed. CAT and POX expressions were analyzed using RT-qPCR with specific primers constructed from the target gene sequences from the Brazilian Coffee Genome Database. Slow drying showed better physiological quality for seeds at 40 and 12% moisture levels, while quick drying was the most effective for seeds with 20% moisture. Sensitivity to water loss was confirmed by quick drying and activation of enzymes. CAT and POX transcriptions reduced during drying. RT-qPCR revealed a complex gene-expression pattern during the oxidative process, with high gene expression in wet seeds.

A heterotrimeric G protein, G alpha i-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epithelial cells.

A heterotrimeric G alpha i subunit, alpha i-3, is localized on Golgi membranes in LLC-PK1 and NRK epithelial cells where it colocalizes with mannosidase II by immunofluorescence. The alpha i-3 was found to be localized on the cytoplasmic face of Golgi cisternae and it was distributed across the whole Golgi stack. The alpha i-3 subunit is found on isolated rat liver Golgi membranes by Western blotting and G alpha i-3 on the Golgi apparatus is ADP ribosylated by pertussis toxin. LLC-PK1 cells were stably transfected with G alpha i-3 on an MT-1, inducible promoter in order to overexpress alpha i-3 on Golgi membranes. The intracellular processing and constitutive secretion of the basement membrane heparan sulfate proteoglycan (HSPG) was measured in LLC-PK1 cells. Overexpression of alpha i-3 on Golgi membranes in transfected cells retarded the secretion of HSPG and accumulated precursors in the medial-trans-Golgi. This effect was reversed by treatment of cells with pertussis toxin which results in ADP-ribosylation and functional uncoupling of G alpha i-3 on Golgi membranes. These results provide evidence for a novel role for the pertussis toxin sensitive G alpha i-3 protein in Golgi trafficking of a constitutively secreted protein in epithelial cells.

Sorting by COP I-coated vesicles under interphase and mitotic conditions.

COP I-coated vesicles were analyzed for their content of resident Golgi enzymes (N-acetylgalactosaminyltransferase; N-acetylglucosaminyltransferase I; mannosidase II; galactosyltransferase), cargo (rat serum albumin; polyimmunoglobulin receptor), and recycling proteins (-KDEL receptor; ERGIC-53/p58) using biochemical and morphological techniques. The levels of these proteins were similar when the vesicles were prepared under interphase or mitotic conditions showing that sorting was unaffected. The average density relative to starting membranes for resident enzymes (14-30%), cargo (16-23%), and recycling proteins (81-125%) provides clues to the function of COP I vesicles in transport through the Golgi apparatus.

Forskolin inhibits and reverses the effects of brefeldin A on Golgi morphology by a cAMP-independent mechanism.

Brefeldin A (BFA) causes rapid redistribution of Golgi proteins into the ER, leaving no definable Golgi apparatus, and blocks transport of proteins into post-Golgi compartments in the cell. In this study we follow the disassembly of the Golgi apparatus in BFA-treated, living cells labeled with NBD-ceramide and demonstrate that forskolin can both inhibit and reverse this process. Long, tubular processes labeled with NBD-ceramide were observed emerging from Golgi elements and extending out to the cell periphery in cells treated with BFA for 5 min. With longer incubations in BFA, the NBD label was dispersed in a fine reticular pattern characteristic of the ER. Treatment with forskolin inhibited these effects of BFA as well as BFA's earliest morphologic effect on the Golgi apparatus: the redistribution to the cytosol of a 110-kD Golgi peripheral membrane protein. In addition, forskolin could reverse BFA's block in protein secretion. Forskolin inhibition of BFA's effects was dose dependent and reversible. High concentrations of BFA could overcome forskolin's inhibitory effect, suggesting forskolin and BFA interact in a competitive fashion. Remarkably, in cells already exposed to BFA, forskolin could reverse BFA's effects causing the 110-kD Golgi peripheral membrane protein to reassociate with Golgi membrane and juxtanuclear Golgi complexes to reassemble. Neither membrane permeant cAMP analogues nor cAMP phosphodiesterase inhibitors could replicate or enhance forskolin's inhibition of BFA. 1,9-Dideoxyforskolin, which does not activate adenylyl cyclase, was equally as effective as forskolin in antagonizing BFA. A derivative of forskolin, 7-HPP-forskolin, that is less potent than forskolin at binding to adenylyl cyclase, was also equally effective as forskolin in antagonizing BFA. In contrast a similar derivative, 6-HPP-forskolin, that is equipotent with forskolin at binding to adenylyl cyclase, did not inhibit BFA's effects. These results suggest that forskolin acts as a competitive antagonist to BFA, using a cAMP-independent mechanism to prevent and reverse the morphologic effects induced by BFA.

Cell type-dependent variations in the subcellular distribution of alpha-mannosidase I and II.

alpha-mannosidases I and II (Man I and II) are resident enzymes of the Golgi complex involved in oligosaccharide processing during N-linked glycoprotein biosynthesis that are widely considered to be markers of the cis- and medial-Golgi compartments, respectively. We have investigated the distribution of these enzymes in several cell types by immunofluorescence and immunoelectron microscopy. Man II was most commonly found in medial- and/or trans- cisternae but showed cell type-dependent variations in intra-Golgi distribution. It was variously localized to either medial (NRK and CHO cells), both medial and trans (pancreatic acinar cells, enterocytes), or trans- (goblet cells) cisternae, or distributed across the entire Golgi stack (hepatocytes and some enterocytes). The distribution of Man I largely coincided with that of Man II in that it was detected primarily in medial- and trans-cisternae. It also showed cell type dependent variations in its intra-Golgi distribution. Man I and Man II were also detected within secretory granules and at the cell surface of some cell types (enterocytes, pancreatic acinar cells, goblet cells). In the case of Man II, cell surface staining was shown not to be due to antibody cross-reactivity with oligosaccharide epitopes. These results indicate that the distribution of Man I and Man II within the Golgi stack of a given cell type overlaps considerably, and their distribution from one cell type to another is more variable and less compartmentalized than previously assumed.

Cholesterol-independent targeting of Golgi membrane proteins in insect cells.

Distinct lipid compositions of intracellular organelles could provide a physical basis for targeting of membrane proteins, particularly where transmembrane domains have been shown to play a role. We tested the possibility that cholesterol is required for targeting of membrane proteins to the Golgi complex. We used insect cells for our studies because they are cholesterol auxotrophs and can be depleted of cholesterol by growth in delipidated serum. We found that two well-characterized mammalian Golgi proteins were targeted to the Golgi region of Aedes albopictus cells, both in the presence and absence of cellular cholesterol. Our results imply that a cholesterol gradient through the secretory pathway is not required for membrane protein targeting to the Golgi complex, at least in insect cells.

Glycosidases of rat Kupffer cells, hepatocytes and peritoneal macrophages.

The acid glycosidase content of rat liver Kupffer cells was compared with that of hepatocytes and resident peritoneal macrophages. Homogenates of all these cells were able to hydrolyze the p-nitrophenyl glycosides of N-acetylglucosamine, N-acetylgalactosamine, glucose, galactose, fucose and mannose, but not xylose. Activity was greatest against the N-acetylglucosaminoside. With Kupffer cell homogenates, most of the glycosidases behaved as if they were lysosomal enzymes. When expressed as rates of hydrolysis per 10(6) cells, activities against a given substrate by homogenates from the three cell types generally agreed within a factor of 2-4. Significant differences between cell types were found, however, when ratios of glycosidase activities were compared. Furthermore, even though the quantity of glycosidase per cell was similar in Kupffer cells and hepatocytes, the glycosidase concentrations were much higher in the former cells, since Kupffer cells are much smaller than hepatocytes.

Regulation of lysosomal and ubiquitin degradative pathways in differentiating human intestinal Caco-2 cells.

The expression of various components of the lysosomal and ubiquitin-dependent degradative pathways was characterized in an in vitro model of differentiating enterocytes, the human colon adenocarcinoma Caco-2 cell line. The activities of the cell-associated lysosomal enzymes alpha-D-mannosidase, beta-hexosaminidase, beta-glucuronidase, and beta-galactosidase increased approximately 2- to 4-fold as differentiation proceeded. In contrast, the protein levels of the two mannose 6-phosphate receptors (MPRs), the insulin-like growth factor II/cation-independent MPR (IGF-II/CI-MPR) and the cation-dependent MPR (CD-MPR), did not change significantly during Caco-2 differentiation. In addition, quantitative Western blot analyses revealed that on a molar basis the CD-MPR is 3.5 times more abundant than the IGF-II/CI-MPR in Caco-2 cells. Since only limited secretion of lysosomal enzymes was observed throughout differentiation, the level of expression of the MPRs was sufficient to target the increased levels of lysosomal enzymes to the lysosome. Unlike the expression of lysosomal enzymes, Western blot analysis demonstrated an approximately 40% and approximately 30% decrease, respectively, in the steady-state levels of free and conjugated ubiquitin during Caco-2 differentiation. Taken together, these results show that the ubiquitin-dependent proteolytic pathway is regulated differently than the lysosomal degradative pathway during Caco-2 differentiation.

The Saccharomyces cerevisiae processing alpha 1,2-mannosidase is localized in the endoplasmic reticulum, independently of known retrieval motifs.

The yeast-specific alpha 1,2-mannosidase, Mns1p, converts Man,GlcNAc2 to a single isomer of Man8GlcNAc2 during N-linked oligosaccharide processing in Saccharomyces cerevisiae. Mns1p is a 68 kDa type II integral membrane glycoprotein with a very short amino terminal cytoplasmic tail of only two amino acids and a large carboxy-terminal catalytic region that is homologous to class 1 alpha 1,2-mannosidases from mammalian and other species. We have used immunofluorescence and immunoelectron microscopy to demonstrate that Mns1p is localized in the endoplasmic reticulum in Saccharomyces cerevisiae. As Mns1p contains none of the known endoplasmic reticulum retrieval motifs (HDEL, KK or RR), these results suggest that Mns1p is localized in the endoplasmic reticulum by a different retentin mechanism.

Golgi proteins persist in the tubulovesicular remnants found in brefeldin A-treated pancreatic acinar cells.

Brefeldin A (BFA) blocks protein export from the endoplasmic reticulum (ER) and causes dismantling of the Golgi cisternae with relocation of resident Golgi proteins to the ER in many cultured cell lines. We examined the effects of BFA on Golgi organization and the distribution of Golgi markers in the rat exocrine pancreas. Immediately after BFA addition, Golgi stacks began to disorganize and Golgi cisternae to vesiculate, and by 15 min no intact Golgi cisternae remained. However, even after prolonged BFA incubation, clusters of small vesicles surrounded by transitional elements of the ER persisted both in the Golgi region and dispersed throughout the apical cytoplasm. These vesicles were morphologically heterogeneous in the density of their content and in the presence of cytoplasmic coats. Immunogold labeling demonstrated that some vesicles within the clusters contained gp58, a cis Golgi marker, and some contained alpha-mannosidase II, a middle/trans Golgi marker in this cell type. Neither marker was detected in the rough ER by immunogold or immunofluorescence labeling. When AlF4- was added during BFA treatment some of the vesicles in the clusters appeared coated. When microsomes were subfractionated into Golgi (light) and rough ER (heavy) fractions on sucrose density gradients, greater than 65% of alpha-mannosidase II and galactosyltransferase activities were found in light fractions (1.14-1.16 g/ml) in both control and BFA-treated lobules. In both cases equally low enzyme activity was recovered in heavier fractions (1.2-1.23 g/ml) containing RNA and alpha-glucosidase activity. However, 5 to 8% of the total recovered RNA consistently codistributed with the Golgi enzyme peak. These results indicate that BFA rapidly inhibits secretion and causes dismantling of the Golgi stacks in pancreatic acinar cells, but clusters of vesicles consisting of bona fide Golgi remnants persist even with prolonged exposure to BFA. Many of the vesicles contain Golgi markers by immunolabeling. By cell fractionation Golgi membrane enzyme activities are recovered in equal amounts in light (Golgi) fractions in both controls and BFA-treated specimens. These findings indicate that in the exocrine pancreas there is a dissociation of BFA's effects on the exocytic pathway: there is a block in transport and Golgi organization is disrupted, but remnant Golgi vesicles and tubules persist and retain Golgi membrane antigens and enzyme activities.

Presence of Golgi remnant membranes in the cytoplasm of brefeldin A-treated cells.

Brefeldin A (BFA) rapidly blocks secretion, induces disassembly of the Golgi complex and causes a redistribution of Golgi components into the endoplasmic reticulum (ER). In addition to these effects on the exocytotic pathway, BFA has been shown to induce fusion of endosomal membranes with the trans-Golgi network in some cell types. To better understand the mechanism through which BFA disrupts the exocytotic traffic, we have examined its effects on the ultrastructural organization of the Golgi complex. Within minutes of exposure to BFA, the Golgi cisternae were fragmented into a number of small tubules and vesicles, many of which had a non-clathrin coat on their cytosolic surface. In addition, a complex structure consisting of anastomosing tubules and associated vesicles appeared in the cytoplasm of cells incubated with BFA for 10 min or longer. These tubular networks were permanent, distinct structures separated from the ER cisternae. They contained cis, middle, and trans Golgi proteins as well as the lipid analogue C5-DMB-ceramide. Furthermore, secretory proteoglycans en route through the Golgi were retained in the lumen of the tubular networks. As judged by the endocytosis of cationized ferritin, endosomes do not contribute to the formation of these tubular networks. Reassembly of the Golgi complex after BFA incubation involved fragmentation and reorganization of the tubular networks as well as fusion with vesicles budded from the ER. We conclude that although in the presence of BFA the bulk of Golgi membranes are induced to fuse with the ER, as indicated by the detection of Golgi markers in this organelle, a fraction of these membranes remain in the cytoplasm organized as Golgi remnants.

Nocodazole and taxol affect subcellular compartments but not secretory activity of GH3B6 prolactin cells.

The effects of two drugs known to affect microtubules (nocodazole, a depolymerizing agent, and taxol, a polymerizing and stabilizing agent) have been tested in GH3B6 prolactin (PRL) cells, a rat pituitary cell line. Under basal condition, GH3B6 cells displayed a dense and entangled microtubule (MT) network, and a tight perinuclear cage of cytokeratin fibers with branching bundles in the cytoplasm. Nocodazole induced a disappearance of MT in the cytoplasm accompanied by the formation of tubulin blebs at the cell periphery, and a slackening of the perinuclear cage of cytokeratin. Taxol induced the formation of straight MT bundles in the cytoplasm, and a tightening of the cytokeratin cage. In parallel, nocodazole induced a fragmentation of the Golgi apparatus which appeared, after staining with antibodies against PRL or against mannosidase II, a Golgi membrane antigen, as small subunits dispersed in the cytoplasm. Taxol induced a perturbation of the Golgi apparatus which, however, remained located near the nucleus. Surprisingly, despite their obvious effects on the subcellular organization, the two MT drugs did not perturb the basal and thyroliberin (TRH)-stimulated PRL release. Moreover, they do not seem to affect the intracellular transport and release of neosynthesized PRL as appreciated by "pulse-chase" experiments. These observations demonstrate that, although MT assume an important role in the spatial compartmentalization of GH3B6 cells, they are not directly involved in the different steps of the intracellular PRL transport from its synthesis site to its release site, as well as in the associated membrane traffic.

Mannosidosis in three brothers--a review of the literature.

Three brothers with mannosidosis were studied, and their clinical and biochemical manifestations are compared with those of 41 cases in the literature. All three boys have psychomotor and growth retardation, characteristic facies, recurrent respiratory infections, sensorineural deafness, craniosynostosis, protuberant abdomens, and thin limbs. Roentgenographic findings of mild dysostosis multiplex, thick calvaria, abnormally contoured vertebrae, coarse trabeculi and thin cortices are consistent with those of reported cases. The lymphocytes of peripheral blood and bone marrow are vacuolated. Alpha-mannosidase deficiency in leukocytes and cultured skin fibroblasts and glycoproteinuria have been documented. The biochemistry of this glycoproteinosis and the pitfalls in diagnosis, such as improper assay conditions of pH and substrate concentration, are discussed. Extrapolation of in vitro and animal model studies suggest that trace metal therapy may be more effective than attempts at enzyme replacement to treat this hereditary storage disease.

Localization of endogenous furin in cultured cell lines.

Furin is a dibasic endopeptidase responsible for the proteolytic maturation of many precursor proteins in the secretory and endocytic pathways of mammalian cells. The levels of furin expression in most cells are very low, and this has hampered attempts to identify the intracellular compartments in which endogenous furin is localized. We have used a specific antibody reagent to a sequence in the carboxy terminus of furin to perform immunofluorescent staining of mammalian cell lines. This antibody was sensitive enough to detect staining for furin in various cell lines. For the most part, furin staining was confined to a juxtanuclear structure characteristic of the Golgi complex. Analyses by video microscopy and confocal microscopy showed that the distribution of furin was distinct from that of mannosidase II, a marker of the Golgi stack, and most closely resembled that of TGN38, a marker of the trans-Golgi network. Therefore, our results suggest that endogenous furin is predominantly localized to the area of the Golgi complex, most likely within the trans-Golgi network.

Identification of glycoprotein storage diseases by lectins: a new diagnostic method.

The specific diagnosis of glycoprotein storage diseases is made by demonstrating a deficiency in enzyme activity or an elevation of undegraded oligosaccharides in cells or body fluids. Prospective sampling and expensive specialized biochemistry, which is also time consuming, are required for such studies. We used lectin reagents on paraffin-embedded tissue sections to identify the specific sugars in undegraded stored substances. We studied 22 cases of glycoprotein storage diseases and differentiated histochemically between alpha- and beta-mannosidosis, fucosidosis, and sialisidosis. Cells affected with alpha-mannosidosis stained with Concanavalia ensiformis (Con A), Triticum vulgaris (WGA), and succinyl-WGA (S-WGA), while beta-mannosidosis cells did not stain with any of the lectins used. In fucosidosis the affected cells stained with Ulex europeus-I (UEA-I), while sialisidosis-affected cells stained with WGA, and in three cases with Arachis hypogea (PNA). This study indicates that lectin histochemistry provides a reliable specific diagnostic pattern for some glycoprotein storage diseases using a simple and inexpensive method.

Alpha-L-fucosidase in cultured bone marrow fibroblasts from fucosidosis patients.

Bone marrow fibroblasts were cultured from two patients with fucosidosis type 2, six control subjects, and three patients with other lysosomal disorders. Optimal conditions for measuring alpha-L-fucosidase activity in lysates of these cells with the fluorogenic substrate 4-methylumbelliferyl-alpha-L-fucoside were established. The pH profile of normal bone marrow fibroblasts showed three peaks and a shoulder of enzymatic activity, with maximum activity at pH 4.75. In cells derived from fucosidosis patients two peaks of apparent alpha-L-fucosidase activity were obtained; the pH optimum was 4.5. alpha-L-Fucosidase activity (mean +/- SD) in the fucosidosis and control bone marrow fibroblasts was 2.5 and 312.4 +/- 10.9 nmoles 4-methylumbelliferone per milligram protein per hour, respectively. A reduction in the apparent specific enzymatic activity in the fucosidosis cells was observed by using increasing concentrations of cellular protein in the assay system. Mixing experiments between normal and fucosidosis cells gave the expected activities. These findings indicate that cultured bone marrow fibroblasts can be used for the diagnosis and study of fucosidosis.

Purification and characterization of beta-mannosidase from human placenta.

Lysosomal beta-mannosidase was purified almost 10,000-fold from human placenta. The final preparation showed several protein bands on polyacrylamide gel electrophoresis. Its molecular mass was estimated to be 110 kDa, the optimal pH was 4.5, the Km was 0.56 mM, and the isoelectric point was 4.7. The enzyme was found to bind completely to Con A-Sepharose, and the pI was not changed after neuraminidase treatment. These results indicate that the purified enzyme represents a lysosomal form which contains high mannose type oligosaccharide chains and only a few sialic acids, if any.

Diagnostic potential of lysosomal hydrolases in body cavity effusions.

Hexosaminidase, alpha-mannosidase, beta-galactosidase, beta-glucuronidase, and arylsulphatase A were measured inperitoneal and pleural effusions from patients with benign, malignant, and inflammatory disorders. Compared with the benign transudates, all enzyme activities were moderately elevated in malignant effusions and markedly elevated in inflammatory effusions. The assay of hexosaminidase and and alpha-mannosidase indicated clearly the underlying pathology in most specimens studied. This method could be of clinical value when the cause of an effusion is in doubt, particularly since the diagnostic criteria are independent of the presence or absence of tumour cells in the aspirate.

The effects of the availability of diet on the levels of exoglycosidases in the supragingival plaque of macaque monkeys.

Supragingival plaque from macaque monkeys was assayed for 13 exoglycosidase enzymes, with appropriate p-nitrophenylglycosides and N-acetylneuramin-lactose used as substrates. Protein in each plaque sample was quantitated with a Coomassie Brilliant Blue dye binding assay, and the specific activity of each enzyme was calculated. In monkeys fed a starch-based diet, fasting resulted in significant increases in the levels of alpha-L-fucosidase, beta-N-acetyl-D-glucosaminidase, beta-N-acetyl-D-galactosaminidase, and neuraminidase. Such changes are consistent with the hypothesis that plaque bacteria degrade salivary glycoproteins for their growth and maintenance in both the presence and especially the absence of dietary food. In contrast, fasting monkeys previously fed a sucrose-rich diet showed no significant alterations in the specific activities of those enzymes whose levels were increased in the starch-based diet group. It is considered likely that, under the conditions prevailing when the sucrose-rich diet is fed, the effective and maximal utilization of sucrose by plaque bacteria necessitates the increased mobilization of nitrogen sources, including the amino sugars of glycoproteins.

Intracellular and extracellular alpha-D-mannosidase activity of cultured skin fibroblasts: relationship to cystic fibrosis.

Fibroblast intracellular alpha-D-mannosidase was inactivated by 30--50% after 120 min at 50 degrees C. Separation of alpha-D-mannosidase by isoelectrofocusing and wheat germ lectin-Sepharose into neutral and acid components showed the former to be heat labile and the cause of the observed loss. Fetal calf serum acid alpha-D-mannosidase could be inactivated at 60 degrees C if kept at pH 9.0. Fibroblast extracellular alpha-D-mannosidase was inactivated by 50--70% after 120 min at 50 degrees C, reflecting the greater level of the neutral component. Control and cystic fibrosis alpha-D-mannosidase showed no difference.