The dynamics of engineered resident proteins in the mammalian Golgi complex relies on cisternal maturation.

After leaving the endoplasmic reticulum, secretory proteins traverse several membranous transport compartments before reaching their destinations. How they move through the Golgi complex, a major secretory station composed of stacks of membranous cisternae, is a central yet unsettled issue in membrane biology. Two classes of mechanisms have been proposed. One is based on cargo-laden carriers hopping across stable cisternae and the other on "maturing" cisternae that carry cargo forward while progressing through the stack. A key difference between the two concerns the behavior of Golgi-resident proteins. Under stable cisternae models, Golgi residents remain in the same cisterna, whereas, according to cisternal maturation, Golgi residents recycle from distal to proximal cisternae via retrograde carriers in synchrony with cisternal progression. Here, we have engineered Golgi-resident constructs that can be polymerized at will to prevent their recycling via Golgi carriers. Maturation models predict the progress of such polymerized residents through the stack along with cargo, but stable cisternae models do not. The results support the cisternal maturation mechanism.

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.

Abnormal expression of acid glycosidases in seminal plasma and spermatozoa from infertile men with varicocele.

The activities of acid beta-glucuronidase, alpha-mannosidase, alpha-glucosidase, alpha-galactosidase, beta-galactosidase and beta-N-acetylglucosaminidase were analysed in seminal plasma and spermatozoa from 26 infertile men with varicocele and from 36 men of normal fertility. Semen samples from ten men with non-obstructive azoospermia were used as control specimens that contained the other components of semen. Spermatozoa were solubilized by both physical (homogenization) and chemical (Triton-X100) methods to obtain the soluble and non-soluble fractions. The activities of several glycosidases measured both in seminal plasma and spermatozoa were directly correlated with the numbers of spermatozoa and sperm motility, confirming previous studies. As some infertile patients with varicocele have normal semen parameters, whereas others have low numbers of spermatozoa and low sperm motility, the varicocele patients were prospectively divided into two groups: one (n = 15) with normal spermiograms and the other (n = 11) with abnormal spermiograms. The activities (expressed in mU ml(-1)) of alpha-mannosidase, beta-galactosidase and beta-N-acetylglucosaminidase in seminal plasma of normozoospermic infertile patients with varicocele were significantly higher than those of fertile controls, but not when expressed in U per 10(8) spermatozoa. The activities of beta-glucuronidase, alpha-mannosidase, beta-galactosidase and beta-N-acetylglucosaminidase in seminal plasma when expressed in U per 10(8) spermatozoa in varicocele patients with abnormal spermiograms were significantly higher than in those of men of normal fertility. The activity of alpha-mannosidase in the soluble fraction of sperm homogenates, expressed as U per 10(8) spermatozoa, was significantly higher in infertile patients with varicocele and abnormal spermiograms than in controls. In the non-soluble fraction of spermatozoa from infertile patients with varicocele, there was an increase in the expression of beta-galactosidase and beta-N-acetylglucosaminidase activities compared with the fraction of spermatozoa from fertile subjects. In summary, infertile patients with varicocele displayed an overexpression of acid alpha-mannosidase, beta-galactosidase and beta-N-acetylglucosaminidase activities in seminal plasma and spermatozoa that may be associated with functional defects in spermatozoa as these glycosidases play an important role in mammalian fertilization.

Polysaccharide analysis using carbohydrate gel electrophoresis: a method to study plant cell wall polysaccharides and polysaccharide hydrolases.

A method to characterize plant cell wall polysaccharides is presented. The complexity of the polymer structures and the large number of different charged and uncharged monosaccharides that make up plant polysaccharides have previously made analysis technically demanding and laborious. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) relies on derivatization of reducing ends of sugars and oligosaccharides with a fluorophore, followed by electrophoresis under optimized conditions in polyacrylamide gels. We show that PACE is a sensitive and simple tool for studying the monosaccharide composition of polysaccharides and of cell wall preparations. In combination with specific hydrolases, it can be used to analyze the structure of polysaccharides. Moreover, the specificity and kinetics of the plant polysaccharide hydrolases themselves can be quickly and effectively studied. PACE can detect as little as 500 fmol of monosaccharides and 100 fmol of oligosaccharides, and it is fast and quantitative.

Gel diffusion assays for endo-beta-mannanase and pectin methylesterase can underestimate enzyme activity due to proteolytic degradation: a remedy.

The accuracy of the sensitive gel-diffusion assay for endo-beta-mannanase activity was improved when protein was added to fruit extracts or into the substrate-gel matrix in which the enzyme assays were conducted. Mixing of commercially available protease inhibitors with fruit enzyme extracts also resulted in increased assayable activity. These treatments were less effective when applied to extracts from tomato seeds, which contained over three times more endogenous protein than fruit extracts. Thus the presence of added or higher amounts of endogenous proteins served as the protectant for endo-beta-mannanase during the course of the gel-diffusion assay, which required an incubation at 32 degrees C for at least 18 h. There was no difference in assayable endo-beta-mannanase activity in the presence and absence of added protein when measured rapidly by viscometry. An effective modification was made to the galactomannan substrate gel assay for endo-beta-mannanase, which is the most efficient method for assaying large numbers of extracts, to improve its accuracy when the enzyme is obtained from tissues containing a low endogenous protein content. This involved incorporating an optimal concentration of gelatin into the galactomannan assay matrix gel. Much higher enzyme activities were recorded, with up to a 10-fold increase for tomato fruit extracts, compared to the same samples assayed on gels with no gelatin added. This increased activity was also obtained using extracts from the fruit of cantaloupe, peach, and nectarine. When incorporated into esterified pectin substrate gels, gelatin also increased the assayable activity of pectin methylesterase. Thus the incorporation of protein (gelatin) into substrate gels during the assay also should be widely more useful for other cell-wall-mobilizing enzymes and hydrolases.

Buds of the Golgi apparatus in parotid acinar cells.

The Golgi apparatus (GA) is a membranous organelle composed of stacked cisterns with associated vesicles. This study was undertaken to determine its origin in rat parotid acinar cells. The morphogenesis of the GA could be recognized in the developmental process as well as in mitotic division of cells. EM studies depicted an aggregation of small vesicles in the early stage of postnatal development or mitosis, that appeared to be the rudimental element of GA. Brefeldin A induced rapid degradation of the cisternal structure to vesicular aggregates. Reconstruction of the GA structure based on these remnant vesicles was observed upon removal of the drug. Similar membranous assembly could be observed after destruction of microtubules. These membranous aggregates presumably corresponded to 'buds of the GA' in parotid acinar cells. However, conventional cytochemical markers for GA were not detected on such immature form of GA. We found that the GA matrix protein GM130 and osmium reductivity (a classical marker for cis-Golgi elements) were consistently localized in the GA elements. Therefore, immunohistochemical distribution of GM130 and osmium impregnation of parotid acinar cells were studied under various dynamic conditions that produced structural modification of the GA.

Enzymatic activity of endophytic fungi of six native seedling species from Doi Suthep-Pui National Park, Thailand.

Endophytic fungi were isolated from the stems, petioles, midribs, and leaves of seedlings of six native tree species collected from Doi Suthep-Pui National Park, Thailand. Endophytes were isolated from all tissue samples investigated, and taxa included five ascomycetes, eight anamorphic taxa, and numerous sterile mycelia. Twenty-six strains were tested for their ability to produce cellulase, mannanase, proteinase, and xylanase. The ability to produce these enzymes was distributed amongst the strains tested. Rainforest seedlings supported a diverse array of endophytes that have a wide range of enzymatic activities. The implication of enzyme production in relation to lifestyle abilities of the endophytes is discussed.

A novel thermostable xylanase from Thermomonospora sp.: influence of additives on thermostability.

An alkalothermophilic Thermomonospora sp. producing high levels of xylanase was isolated from self-heating compost. The culture produced 125 IU/ml of xylanase when grown in shake flasks at pH 9 and 50 degrees C for 96 h. The culture filtrate also contained cellulase (23 IU/ml), mannanase (1 IU/ml) and beta-xylosidase (0.1 IU/ml) activities. The xylanase was active at a broad range of pH (5-9) and temperature (40-90 degrees C). The optimum pH and temperature were 7 and 70 degrees C, respectively. The enzyme was stable in the pH range 5-8 and was thermostable with half-lives of 8 and 4 h at 60 degrees C and 70 degrees C, respectively, but only 9 min at 80 degrees C. The effects of a variety of compounds to enhance the stability of xylanase at 80 degrees C was studied. Addition of sorbitol, mannitol and glycerol increased the thermostability of xylanase in proportion to the number of hydroxyl groups per polyol molecule. Glycine also offered protection against thermoinactivation. Xylan, trehalose, gelatin and trehalose-gelatin mixture had marginal effect on the thermostability of xylanase at 80 degrees C.

N-ethylmaleimide-sensitive factor is associated with the nuclear envelope.

N-Ethylmaleimide-sensitive factor (NSF) is an ATPase involved in many membrane fusion events within the exocytic and endocytotic pathways. In the present study we showed that NSF is associated with the nuclear envelope. Golgi-associated NSF was released from membranes upon incubation with Mg(2+)-ATP, reflecting the disassembly of a complex consisting of NSF, soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). In contrast nuclear envelope-associated NSF in interphase cells was not released by the same treatment. During mitosis, however, it was released from nuclear membranes by Mg(2+)-ATP. These results suggest that the binding mode of nuclear membrane-associated NSF changes during the cell cycle.

Production and properties of hemicellulases by a Thermomyces lanuginosus strain.

Thermophilic fungi producing extremely high beta-xylanase and their associated hemicellulases have attracted considerable attention because of potential industrial applications. Thermomyces lanuginosus strain SSBP isolated from soil, produced beta-xylanase activity of 59 600 nkat ml-1 when cultivated on a medium containing corn cobs as substrate and yeast extract as nitrogen source. Lower beta-xylanase activities were produced after growth on other xylan substrates, sugars and soluble starch. Other hemicellulases were produced extracellularly at significantly lower levels than the beta-xylanase activity produced on corn cobs. No cellulase activity was observed. The optimal conditions for beta-xylanase production were 50 degrees C and pH 6.5, whereas 70 degrees C and between pH 5. 5 and 9.5 were optimal for beta-xylanase activity. The temperature optima for other hemicellulases were less than the xylanase with the exception of beta-mannosidase. The pH optima of the other hemicellulases were between 5.0 and 6.5. Xylanase was stable up to 70 degrees C and between pH 5.5 and 9.0 for 30 min whereas the other hemicellulase were less stable. These results suggest that the most suitable conditions for hydrolysis of hemicellulose by these enzymes would be at 50 degrees C and pH 6.0.

Detecting mannosidase activities using ribonuclease B and matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Ribonuclease (RNase) B incubated with purified enzymes, whole bacterial cultures, or their separated components-cells and supernates-have been directly analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-ToF) to detect exomannosidases and to evaluate their specificities and location. Enzymatic cleavage was monitored by observing changes in RNase B glycoform population. Thus a nonspecific alpha-(1 --> 2)-mannosidase activity converts the glycoprotein to its Man(5) form, identifiable by its mass of 14,899 [M + H](+); this species subsequently is converted, by the actions of alpha-(1 --> 3) and alpha-(1 --> 6)-mannosidases, to the Man(1) form via Man(4), Man(3), and Man(2). The Man(1) glycoform (which is readily isolated) has then similarly been used for identifying beta-(1 --> 4)-mannosidase and the derived Man(0) form has served in turn as a natural substrate for beta-(1 --> 4) N-acetylglucosaminidase producing a species possessing a single asparagine-linked GlcNAc residue (mass 13,886). Mannose liberated from the actions of mannosidases can, if desired, be quantified by, for example, chromatography. The actions and specificities of endoglycosidases such as a peptide-N-glycosidase F (PNGase F) and of endo-N-acetlyglucosaminidases (e.g., endo-F and endo-H), which respectively cleave between the GlcNAc&bond;Asn and GlcNAc&bond;GlcNAc bonds of N-linked glycoproteins, are also demonstrable by MALDI-ToF analysis of RNase B (and derived products). From these digests the completely deglycosylated polypeptide corresponding to RNase A in which Asn has been converted to Asp (mass 13,684) and a species corresponding to RNase A + GlcNAc (mass 13,886) are produced, together with their corresponding free oligosaccharides which are amenable to analysis by both MALDI-ToF and by HPLC.

Immunohistochemical evaluation of endomannosidase distribution in rat tissues: evidence for cell type-specific expression.

Asparagine-linked oligosaccharides of glycoproteins are subject to a series of trimming reactions by glucosidases and mannosidases in the endoplasmic reticulum which result in the removal of all three glucose residues and several of the nine mannose residues. At present, endomannosidase represents the only processing enzyme which cleaves internally and provides an alternate deglucosylation pathway. However, in contrast to the endoplasmic reticulum residential proteins glucosidase I and II, endomannosidase is primarily situated in the Golgi apparatus of rat liver hepatocytes and hepatocyte cell lines. We have performed a confocal immunohistochemical study to investigate endomannosidase in various rat tissues and used a monoclonal antibody against Golgi mannosidase II as a marker for the Golgi apparatus. Although immunofluorescence for both endomannosidase and Golgi mannosidase II was detectable in the epithelia of many tissues, renal proximal tubular cells, cortex and medulla of adrenal gland, gastric mucosa, and Leydig cells of testis were unreactive for endomannosidase. Furthermore, the endothelia in all studied tissues were unreactive for endomannosidase but positive for Golgi mannosidase II. It is concluded that by immunohistochemistry endomannosidase exhibits a cell type-specific expression in rat tissues.

The Drosophila GMII gene encodes a Golgi alpha-mannosidase II.

In this paper we show the organisation of the Drosophila gene encoding a Golgi alpha-mannosidase II. We demonstrate that it encodes a functional homologue of the mouse Golgi alpha-mannosidase II. The Drosophila and mouse cDNA sequences translate into amino acid sequences which show 41% identity and 61% similarity. Expression of the Drosophila GMII sequence in CHOP cells produces an enzyme which has mannosidase activity and is inhibited by swainsonine and by CuSO(4.) In cultured Drosophila cells and in Drosophila embryos, antibodies raised against a C-terminal peptide localise this product mainly to the Golgi apparatus as identified by cryo-immuno electron microscopy studies and by antibodies raised against known mammalian Golgi proteins. We discuss these results in terms of the possible use of dGMII as a Drosophila Golgi marker.

Clofibrate inhibits membrane trafficking to the Golgi complex and induces its retrograde movement to the endoplasmic reticulum.

Insights into the function of the Golgi complex have been provided by experiments performed with various inhibitors of membrane trafficking, such as the macrocyclic lactone brefeldin A (BFA), a compound that inhibits constitutive secretion, prevents the formation of coatomer-coated transport vesicles, and stimulates the retrograde movement of Golgi resident enzymes back to the ER. We show here that the structurally unrelated compound clofibrate, a peroxisome proliferator (PP) and hypolipidemic agent, also reversibly disrupts the morphological and functional integrity of the Golgi complex in a manner similar to BFA. In the presence of clofibrate, the forward transport of newly synthesized secretory proteins from the ER to the Golgi is dramatically inhibited. Moreover, clofibrate causes Golgi membranes to travel rapidly in a microtubule-dependent manner back to the ER, forming a hybrid ER-Golgi tubulovesicular membrane network. These affects appear to be independent of clofibrate's ability to stimulate the PP-activated receptor (PPAR) alpha pathway because other PPAR stimulators (DEHP, WY-14643) did not alter the Golgi complex or induce retrograde trafficking. These data suggest that PPAR alpha-independent, clofibrate-sensitive proteins participate in regulating Golgi-to-ER retrograde membrane transport, and, equally importantly, that clofibrate may be used as a pharmacological tool for investigating Golgi membrane dynamics.

A novel Rab GTPase, Rab33B, is ubiquitously expressed and localized to the medial Golgi cisternae.

Small GTP-binding proteins of the Rab family play important roles at defined steps of vesicular transport in protein secretion and the endocytosis pathway. In mammals, more than 30 proteins belonging to the Rab family have been reported to date. We report here the molecular cloning and characterization of a novel Rab protein, Rab33B. The amino acid sequence of Rab33B shows 55.3% identity to the Rab33A protein (previously called S10), and these two proteins share unique amino acid sequences at the effector domain. The genomic organization of rab33B was the same as rab33A: it consists of two exons. Thus, these two proteins make a subclass within the Rab family. Northern blot analysis showed that rab33B is expressed ubiquitously in mouse tissues, in contrast to rab33A whose expression is restricted to the brain and the immune system. A 26 kDa protein was detected by western blotting using a Rab33B-specific monoclonal antibody. Using immunofluorescence studies, Rab33B was shown to co-localize with (alpha)-mannosidase II, a Golgi-specific marker. Immunoelectron microscopy analysis further defined the localization of Rab33B to the medial Golgi cisternae. These results suggest Rab33B plays a role in intra-Golgi transport.

Regulation of the collagenase-3 receptor and its role in intracellular ligand processing in rat osteoblastic cells.

We have previously described a specific, saturable receptor for rat collagenase-3 in the rat osteosarcoma cell line, UMR 106-01. Binding of rat collagenase-3 to this receptor is coupled to the internalization and eventual degradation of the enzyme and correlates with observed extracellular levels of the enzyme. In this study we have shown that decreased binding, internalization, and degradation of 125I-rat collagenase-3 were observed in cells after 24 h of parathyroid hormone treatment; these activities returned to control values after 48 h and were increased substantially (twice control levels) after 96 h of treatment with the hormone. Subcellular fractionation studies to identify the route of uptake and degradation of collagenase-3 localized intracellular accumulation of 125I-rat collagenase-3 initially in Golgi-associated lysosomes and later in secondary lysosomes. Maximal lysosomal accumulation of the radiolabel and stimulation of general lysosomal activity occurred after 72 h of parathyroid hormone treatment. Preventing fusion of endosomes with lysosomes (by temperature shift, colchicine, or monensin) resulted in no internalized 125I-collagenase-3 in either lysosomal fraction. Treatment of UMR cells with the above agents or ammonium chloride decreased excretion of 125I-labeled degradation products of collagenase-3. These experiments demonstrated that degradation of collagenase-3 required receptor-mediated endocytosis and sequential processing by endosomes and lysosomes. Thus, parathyroid hormone regulates the expression and synthesis of collagenase-3 as well as the abundance and functioning of the collagenase-3 receptor and the intracellular degradation of its ligand. The coordinate changes in the secretion of collagenase-3 and expression of the receptor determine the net abundance of the enzyme in the extracellular space.

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.