An improved chemical fixation method suitable for immunogold localization of green fluorescent protein in the Golgi apparatus of tobacco Bright Yellow (BY-2) cells.

In plant systems, the green fluorescent protein (GFP) is increasingly used as a marker to study dynamics of the secretory apparatus using fluorescence microscopy. The purpose of this study was to immunogold localize the GFP, at the electron microscopic level, in a line of tobacco BY-2-cultured cells, expressing a GFP-tagged Golgi glycosyltransferase. To this end we have developed a simple, one-step chemical fixation method that allow good structural preservation and specific labeling with anti-GFP antibodies. Using this method, we have been able to show that an N-glycan GFP-tagged xylosyltransferase is specifically associated with Golgi stacks of BY-2 transformed cells and is preferentially located in medial cisternae. As an alternative to cryofixation methods, such as high-pressure freezing, which requires specialized and expensive equipment not available in most laboratories, this method offers researchers the opportunity to investigate GFP-tagged proteins of the endomembrane system in tobacco BY-2 cells.     

Purification, characterization, cDNA cloning and nucleotide sequencing of a cellulase from the yellow-spotted longicorn beetle, Psacothea hilaris.

A cellulase (endo-beta-1,4-glucanase, EC 3.2.1.4) was purified from the gut of larvae of the yellow-spotted longicorn beetle Psacothea hilaris by acetone precipitation and elution from gels after native PAGE and SDS/PAGE with activity staining. The purified protein formed a single band, and the molecular mass was estimated to be 47 kDa. The purified cellulase degraded carboxymethylcellulose (CMC), insoluble cello-oligosaccharide (average degree of polymerization 34) and soluble cello-oligosaccharides longer than cellotriose, but not crystalline cellulose or cellobiose. The specific activity of the cellulase against CMC was 150 micro mol.min-1.(mg protein)-1. TLC analysis showed that the cellulase produces cellotriose and cellobiose from insoluble cello-oligosaccharides. However, a glucose assay linked with glucose oxidase detected a small amount of glucose, with a productivity of 0.072 micro mol.min-1.(mg protein)-1. The optimal pH of P. hilaris cellulase was 5.5, close to the pH in the midgut of P. hilaris larvae. The N-terminal amino-acid sequence of the purified P. hilaris cellulase was determined and a degenerate primer designed, which enabled a 975-bp cDNA clone containing a typical polyadenylation signal to be obtained by PCR and sequencing. The deduced amino-acid sequence of P. hilaris cellulase showed high homology to members of glycosyl hydrolase family 5 subfamily 2, and, in addition, a signature sequence for family 5 was found. Thus, this is the first report of a family 5 cellulase from arthropods.     

Large scale purification of myo-inositol monophosphate phosphatase from porcine brains

myo-Inositol monophosphate phosphatase (IMPase) has been purified 888-fold to apparent homogeneity from procine brains. The purification procedure involves: homogenization, ammonium sulfate fractionation, and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 932 nmol . min(-1) . mg(-1). The molecular mass of the enzyme was estimated to be 29kDa by SDS poly-acrylamide gel electrophoresis and 58 +/- 5 kDa by HPLC gel filtration in 10mM Tris-HCI, pH 7.4. Kinetic measurements have shown that the apparent K(m) value of the phosphatase for the utilization of inositol-1-phosphate and beta-glycerol phosphate are 3.20 x 10(-4) and 8 x 10(-3) M, respectively. Similar to the same enzyme isolated from bovine brains, the porcine brain enzyme has been shown to be inhibited by lithium. The K(1) was determined to be 6.38 x 10(-4) M and the inhibition is uncompetitive. (c) 1995 John Wiley & Sons, Inc.     

An energy-conserving pyruvate-to-acetate pathway in Entamoeba histolytica. Pyruvate synthase and a new acetate thiokinase.

Under anaerobic conditions, cells of Entamoeba histolytica grown with bacteria produce H2 and acetate while cells grown axenically produce neither. Aerobically, acetate is produced and O2 is consumed by amebae from either type of cells. Centrifuged extracts, 2.4 x 106 x g x min, from both types of cells contain pyruvate synthase (EC 1.2.7.1) and an acetate thiokinase which, together, form a system capable of converting pyruvate to acetate. Pyruvate synthase catalyzes the reaction: pyruvate + CoA leads to CO2 + acetyl-CoA + 2E. Electron acceptors which function with this enzyme are FAD, FMN, riboflavin, ferredoxin, and methyl viologen, but not NAD or NADP. The amebal acetate thiokinase catalyzes the reaction acetyl-CoA + ADP + Pi leads to acetate + ATP + CoA. For this apparently new enzyme we suggest the trivial name acetyl-CoA-synthetase (ADP-forming). Extracts from axenic amebae do not contain hydrogenase, but extracts from cells grown with bacteria do. It is postulated that in bacteria-grown amebae electrons generated at the pyruvate synthase step are utilized anaerobically to produce H2 via the hydrogenase and that the acetyl-CoA is converted to acetate in an energy-conserving step catalyzed by amebal acetyl-CoA synthetase. Aerobically, cells grown under either regimen may utilize the energy-conserving pyruvate-to-acetate pathway since O2 then serves as the ultimate electron acceptor.     

Capillaries of the adrenal cortex possess aminopeptidase A and angiotensin-converting-enzyme activities.

The enzymes required to convert the prohormone angiotensin I into angiotensins II and III, secretagogues of aldosterone, are enriched in association with capillary endothelium isolated from rat adrenal cortex. Thus the secretion of aldosterone may be controlled, in part, by processing of peptides occurring within the adrenal gland itself.     

The use of short-term tests to measure the preventive action of reducing agents on formation and activation of carcinogenic nitroso compounds

The effect of reducing agents on the nitrosation of methylguanidine (MG) and on the in vitro activation of dimethylnitrosamine (DMN) was examined by measuring DNA-repair synthesis (unscheduled incorporation of [3h]TdR), shifts in alkaline sucrose gradients, frequency of chromosome aberrations, and clone-forming capacity of cultured human fibroblasts. The reducing agents examined were sodium ascorbate, cysteine, cysteamine, and propyl gallate. Since the short-term bioassays used can be quantitated, it has become relatively easy to detect the inhibitory action of reducing compounds on the nitrosation reaction of MG and metabolic activation (with S-9 preparation) of the precarcinogen DMN, to measure their effective dose range, and to establish the most effective ratios between inhibitory agent and reactant. The results indicate that DNA-repair synthesis is a suitable short-term test for studying the numerous combinations and premutations between several carcinogenic or non-carcinogenic agents, and for estimating the capacity of inhibitory agents to affect formation and activation of chemical carcinogens.     

The use of short-term tests to measure the preventive action of reducing agents on formation and activation of carcinogenic nitroso compounds

The effect of reducing agents on the nitrosation of methylguanidine (MG) and on the in vitro activation of dimethylnitrosamine (DMN) was examined by measuring DNA-repair synthesis (unscheduled incorporation of [³H]TdR), shifts in alkaline sucrose gradients, frequency of chromosome aberrations, and clone-forming capacity of cultured human fibroblasts. The reducing agents examined were sodium ascorbate, cysteine, cysteamine, and propyl gallate. Since the short-term bioassays used can be quantitated, it has become relatively easy to detect the inhibitory action of reducing compounds on the nitrosation reaction of MG and metabolic activation (with S-9 preparation) of the precarcinogen DMN, to measure their effective dose range, and to establish the most effective ratios between inhibitory agent and reactant. The results indicate that DNA-repair synthesis is a suitable short-term test for studying the numerous combinations and permutations between several carcinogenic or non-carcinogenic agents, and for estimating the capacity of inhibitory agents to affect formation and activation of chemical carcinogens.