Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid

Carnivorous plants primarily use aspartic proteases during digestion of captured prey. In contrast, the major endopeptidases in the digestive fluid of the Venus flytrap (Dionaea muscipula) are cysteine proteases (dionain-1 to -4). Here, we present the crystal structure of mature dionain-1 in covalent complex with inhibitor E-64 at 1.5 Å resolution. The enzyme exhibits an overall protein fold reminiscent of other plant cysteine proteases. The inactive glycosylated pro-form undergoes autoprocessing and self-activation, optimally at the physiologically relevant pH value of 3.6, at which the protective effect of the pro-domain is lost. The mature enzyme was able to efficiently degrade a Drosophila fly protein extract at pH 4 showing high activity against the abundant Lys- and Arg-rich protein, myosin. The substrate specificity of dionain-1 was largely similar to that of papain with a preference for hydrophobic and aliphatic residues in subsite S₂ and for positively charged residues in S₁. A tentative structure of the pro-domain was obtained by homology modeling and suggested that a pro-peptide Lys residue intrudes into the S₂ pocket, which is more spacious than in papain. This study provides the first analysis of a cysteine protease from the digestive fluid of a carnivorous plant and confirms the close relationship between carnivorous action and plant defense mechanisms.     

Eco-efficiency indicator framework implemented in the metallurgical industry: part 2—a case study from the copper industry

Purpose

Sustainability differentiation has become an important issue for companies throughout the value chain. There is thus a need for detailed and credible analyses, which show the current status and point out where improvements can be done and how. The study describes how a comprehensive product-centric eco-efficiency indicator framework can be used to evaluate, benchmark, and communicate the sustainability of a copper production value chain. The indicator framework, together with the suggested data collection and simulation methods, aims at evaluating the whole system, while still enabling a focus on scopes of different width. The status of the environment, current production technologies, location-specific and process-specific issues all play a role in achieving sustainable development.

Methods

Copper cathode production from copper ore was chosen to exemplify the developed framework. Data sets from a simulation tool were used when available and LCI databases and LCA software were utilized for the remaining steps. The value chain is analyzed and the benchmark for each indicator built according to the new Gaia Refiner indicator framework. This method enables analysis of specific production steps with a higher degree of accuracy.

Results and discussion

The case study shows how some important environmental sustainability issues in copper production can be analyzed and benchmarked within a product group. Benchmark data is collected and used in the analysis for the selected system scope. Data availability is still an issue and the example shows which areas require more information in this context so that products and value chains can be benchmarked in the future on a more consistent basis. The energy mix, chemical use, and land use contribute to potential environmental sustainability risks within the product benchmarking group, while emissions control shows competitive environmental sustainability advantages for the case study.

Conclusions

The methodology is shown to work well in highlighting the sustainability advantages and risks of value chains in copper production with the selected system scope in a visual manner through the Sustainability Indicator “Flower.” The importance of a baseline is clear. The effect of the metal ore grade on the results shows that the scalability of the analysis system is very important. Scaling the system scope up will show the differences in varying value chains and scaling the system scope down will show efficiency differences between more similar value chains, thus visualizing where innovation has the biggest impact.

     

Enzyme enhancement activity of N-octyl-beta-valienamine on beta-glucosidase mutants associated with Gaucher disease

Gaucher disease (GD), caused by a defect of beta-glucosidase (beta-Glu), is the most common form of sphingolipidosis. We have previously shown that a carbohydrate mimic N-octyl-beta-valienamine (NOV), an inhibitor of beta-Glu, could increase the protein level and enzyme activity of F213I mutant beta-Glu in cultured GD fibroblasts, suggesting that NOV acted as a pharmacological chaperone to accelerate transport and maturation of this mutant enzyme. In the current study, NOV effects were evaluated in GD fibroblasts with various beta-Glu mutations and in COS cells transiently expressing recombinant mutant proteins. In addition to F213I, NOV was effective on N188S, G202R and N370S mutant forms of beta-Glu, whereas it was ineffective on G193W, D409H and L444P mutants. When expressed in COS cells, the mutant proteins as well as the wild-type protein were localized predominantly in the endoplasmic reticulum and were sensitive to Endo-H treatment. NOV did not alter this localization or Endo-H sensitivity, suggesting that it acted in the endoplasmic reticulum. Profiling of N-alkyl-beta-valienamines with various lengths of the acyl chain showed that N-dodecyl-beta-valienamine was as effective as NOV. These results suggest a potential therapeutic value of NOV and related compounds for GD with a broad range of beta-Glu mutations.     

Pseudanthias calloura (Teleostei: Perciformes), a new serranid fish from Palau, Central Pacific

A new anthine fish, Pseudanthias calloura, is described on the basis of three specimens from Palau. The new species differs from other congeners in having a complicated color pattern of the caudal fin and the following characters: moderately forked caudal fin; dorsal fin with 10 spines, the anterior ones being shorter than the rest, and with 16 soft rays; second anal spine shorter than the third; 19 pectoral rays; scales on lateral line 51 (holotype) to 53 (paratypes) in number; tongue without teeth; circumorbital rim with several fleshy projection on posterior part; a row of fine scales occurring asymmetrically behind alternate dorsal spines on both left and right sides; preopercle margin finely serrated; interopercle and subopercle margins smooth. Received: June 9, 1999 / Revised: September 23, 2000 / Accepted: February 23, 2001     

Identification of a novel bile acid in swans, tree ducks, and geese: 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid

By HPLC, a taurine-conjugated bile acid with a retention time different from that of taurocholate was found to be present in the bile of the black-necked swan, Cygnus melanocoryphus. The bile acid was isolated and its structure, established by (1)H and (13)C NMR and mass spectrometry, was that of the taurine N-acyl amidate of 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid. The compound was shown to have chromatographic and spectroscopic properties that were identical to those of the taurine conjugate of authentic 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid, previously synthesized by us from ursodeoxycholic acid. By HPLC, the taurine conjugate of 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid was found to be present in 6 of 6 species in the subfamily Dendrocygninae (tree ducks) and in 10 of 13 species in the subfamily Anserinae (swans and geese) but not in other subfamilies in the Anatidae family. It was also not present in species from the other two families of the order Anseriformes. 3alpha,7alpha,15alpha-Trihydroxy-5beta-cholan-24-oic acid is a new primary bile acid that is present in the biliary bile acids of swans, tree ducks, and geese and may be termed 15alpha-hydroxy-chenodeoxycholic acid.     

Kinematics and kinetics of the racket-arm during the soft-tennis smash under match conditions

The purposes of this study were to (a) describe the racket-arm kinematics and kinetics of the soft-tennis smash during match rallies, and (b) assess the characteristics of this smash vs. the laboratory-simulated smash of our previous study. In the current study we recorded soft-tennis smash motions during match play of the 3rd East Asian Games. Racket-arm anatomical joint angular velocity and anatomical joint torque were calculated from 3-D coordinate data of 13 collected motions obtained using the direct linear transformation procedure. The results showed that most of the maximum values of the anatomical joint torques were qualitatively smaller than those of the tennis serve. Peak elbow extension, shoulder internal rotation, and elbow varus torques in match play were significantly greater than values reported for laboratory-simulated conditions. The greater forward swing torques did not result in significantly different racket head velocity, possibly because there was a significantly shorter forward swing phase in match conditions. In particular, a clear peak of the elbow extension torque during the forward swing phase was the most characteristic pattern in the smashes under match conditions, for it was 160% greater than laboratory-simulated conditions. These results supported our hypothesis that racket-arm kinematic and kinetic characteristics of the smash under match conditions differ from those under laboratory-simulated conditions. Possible explanations include the time-pressure conditions of the competitive situation in a match, and the Hawthorne effect (Hudson et al., 1986), both of which alter performance between match conditions and laboratory-simulated conditions.     

Akt is an endogenous inhibitor toward tumor necrosis factor-related apoptosis inducing ligand-mediated apoptosis in rheumatoid synovial cells

Akt is known to be activated in the rheumatoid synovial tissues. We examined here functional role of Akt during tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis in rheumatoid synovial cells. Rheumatoid synovial cells in vitro were rapidly committed to apoptosis in response to TRAIL in mitochondria-dependent manner whereas Akt and extracellular signal-regulated kinase (ERK) were also phosphorylated. TRAIL-mediated apoptosis in synovial cells was significantly increased through inactivation of Akt by LY294002, however, that process was not so changed by adding ERK inhibitor, PD98059. Platelet-derived growth factor (PDGF) clearly phosphorylated both Akt and ERK in synovial cells, and PDGF pretreatment markedly suppressed TRAIL-mediated synovial cell apoptosis. The use of not PD98059 but LY294002 abrogated PDGF-mediated inhibitory effect toward TRAIL-induced apoptosis in synovial cells. The above protective effect of Akt was confirmed by the use of short interfering RNA (siRNA)-directed inhibition of Akt. Our data suggest that Akt is an endogenous inhibitor during TRAIL-mediated synovial cell apoptotic pathway, which may explain that synovial cells in situ of the rheumatoid synovial tissues are resistant toward apoptotic cell death in spite of death receptor expression.