Solubilization of cellulosomal cellulases by fusion with cellulose-binding domain of noncellulosomal cellulase engd from Clostridium cellulovorans

Clostridium cellulovorans produces a cellulase complex (cellulosome) as well as noncellulosomal cellulases. In this study, we determined a factor that affected the solubility of the cellulosomal cellulase EngB and the noncellulosomal EngD when they were expressed in Escherichia coli. The catalytic domains of EngB and EngD formed inclusion bodies when expressed in E. coli. On the other hand, both catalytic domains containing the C-terminal cellulose-binding domain (CBD) of EngD were expressed in soluble form. Fusion with the CBD of EngD also helped increased the solubility of cellulosomal cellulase EngL upon expression in E. coli. These results indicate that the CBD of EngD plays an important role in the soluble expression of the catalytic domains of EngB, EngL, and EngD. The possible mechanisms of solubilization by fusion of the catalytic domain with the CBD from EngD are discussed.     

Reduction of glycosphingolipid levels in lipid rafts affects the expression state and function of glycosylphosphatidylinositol-anchored proteins but does not impair signal transduction via the T cell receptor

Lipid rafts are highly enriched in cholesterol and sphingolipids. In contrast to many reports that verify the importance of cholesterol among raft lipid components, studies that address the role of sphingolipids in raft organization and function are scarce. Here, we investigate the role of glycosphingolipids (GSLs) in raft structure and raft-mediated signal transduction in T lymphocytes by the usage of a specific GSL synthesis inhibitor, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Surface GM1 expression and the expression of GSLs in rafts were profoundly reduced by D-PDMP treatment, whereas the expression of other lipid and protein constituents, such as cholesterol, sphingomyelin, Lck, and linker for activation of T cells, was not affected. T cell receptor-mediated signal transduction induced by antigen stimulation or by antibody cross-linking was normal in D-PDMP-treated T cells. In contrast, the signal through glycosylphosphatidylinositol (GPI)-anchored proteins was clearly augmented by D-PDMP treatment. Moreover, GPI-anchored proteins became more susceptible to phosphatidylinositol-specific phospholipase C cleavage in D-PDMP-treated cells, demonstrating that GSL depletion from rafts primarily influences the expression state and function of GPI-anchored proteins. Finally, by comparing the effect of D-PDMP with that of methyl-beta-cyclodextrin, we identified that compared with cholesterol depletion, GSL depletion has the opposite effect on the phosphatidylinositol-specific phospholipase C sensitivity and signaling ability of GPI-anchored proteins. These results indicate a specific role of GSLs in T cell membrane rafts that is dispensable for T cell receptor signaling but is important for the signal via GPI-anchored proteins.     

Formation of Protoplasts from Cultured Tobacco Cells and Arabidopsis thaliana by the Action of Cellulosomes and Pectate Lyase from Clostridium cellulovorans

The crude culture supernatants from Clostridium cellulovorans were tested for their ability to convert plant cells to protoplasts. The supernatants readily released protoplasts from cultured tobacco cells and Arabidopsis thaliana. The crude culture supernatant from pectin-grown cells was more active than supernatants from glucose-, cellobiose-, xylan-, and locust bean gum-grown cells. After removal of cellulosomes, the crude culture supernatant lost its protoplast formation activity. The protoplast formation activity of the crude culture supernatant from C. cellulovorans was more effective than those of commercial enzymes based on protein content.     

Effects of exercise on rats subjected to tail suspension

Deterioration of vital functions is observed in living organisms in microgravity. However, this deterioration can be arrested by adequate exercises. In this study, utilizing tail suspension in rats to simulate microgravity, we examined the influence of running exercise every other day on blood constituents, visceral parameters, and muscles.     

Determination of horserdish peroxidase concentration using the chemiluminescence of Cypridina luciferin analogue, 2 methyl-6-(p-methyoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one

The chemiluminescence of the Cypridina luciferin analogue, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA) was observed at 462nm in the presence of horseradish peroxidase (HRP) and the total spectrum of light emitted was found to depend linearly on HRP concentration. Methods for the determination of HRP concentration using the chemiluminescence was investigated. HRP could be detected in the range from 100 pmol/L to 100nmol/L under the optimum condition, H₂O₂ (10mmol/L) and MCLA (10μmol/L) at pH 5.8.     

A method using fibrin-fixed Blue Dextran for determining the plasmin and plasmin inhibitor activities in human plasma.

The fibrinolytic activity of plasmin was determined by incubating with fibrin-fixed Blue Dextran as a substrate, the Blue Dextran released being proportional to the plasmin activity. The applicability of this method for rapid and accurate evaluation of fibrinolytic activity was demonstrated by dose-response curves with purified plasmin, plasmin generated by urokinase in human plasma and euglobulin. The method can also be used to determined plasmin inhibitors in plasma.     

Affinity chromatography of lipase with hydrophobic ligands coupled to cyanogen bromide-activated agarose.

The behavior of lipase produced by Pseudomans mephitica var. lipopytica toward hydrophobic residues coupled to spacer gels that were prepared by coupling a primary amine to CNBr-activated agarose, was studied. The lipase adsorbed on the ligand of a long unbranched aliphatic chain, a benzene ring, or deoxycholic acid was only slightly or not all eluted at pH 5 or pH 11 by buffers containing 1 M NcC1. The lipase was eluted by liquid containing a surfactant or an organic solvent miscible with water, indicating greater involvement of hydrophobic forces. The adsorption of propane, cyclopentane, cyclohexane, cycloheptane, or chrysene appears to be achieved through electrostatic forces, inasmuch as desorption was caused by buffer containing 1 M NaC1 at pH 11. The amount of lipase adsorbed on these hydrophobic ligands was about the same as that adsorbed on the ligands belonging to the first group. Since little lipase wad adsorbed on cyclopropane, cycloctane, pyridine, methane, n-pentane, or branched aliphatic chains, these ligands appear to impose steric hindrance on the adsorption of lipase, or they may be too small to fit into the hydrophobic sites of lipase.     

A TSH/dibutyryl cAMP activated Cl-/I- channel in FRTL-5 cells.

An iodide (I) and chloride (Cl) channel has been identified in the continuously cultured FRTL-5 thyroid cell line using a cell attached patch clamp technique. The channel is activated by TSH and dibutyryladenosine cyclic monophosphate (Bt2-cAMP) but not by phorbol 12-myristate 13-acetate (TPA). Gluconate can not replace chloride or iodide and the channel is impermeable to Na+,K+ and tetraethylammonium ions. The current-voltage relationship demonstrates that the single channel current is a linear function of the clamp voltage. Single channel currents reversed at a pipette potential close to 0 mV. The mean single channel conductance was 60 pS for Cl- and 50 pS for I-. From the I-V relationship there was a strong outward rectification with Cl-, and a complete block with I-, in the single channel current above +40 mV. The feature of the channel is manifested in the single channel records by four distinct, equally spaced conductance levels. We suggest the channel is important for the transport of I and Cl ions across the apical membrane into the colloid space and is important for hormone synthesis and follicle formation.