Class sigma glutathione transferase unfolds via a dimeric and a monomeric intermediate: impact of subunit interface on conformational stability in the superfamily

Solvent-induced equilibrium unfolding of a homodimeric class sigma glutathione transferase (GSTS1-1, EC 2.5.1.18) was characterized by tryptophan fluorescence, anisotropy, enzyme activity, 8-anilino-1-naphthalenesulfonate (ANS) binding, and circular dichroism. Urea induces a triphasic unfolding transition with evidence for two well-populated thermodynamically stable intermediate states of GSTS1-1. The first unfolding transition is protein concentration independent and involves a change in the subunit tertiary structure yielding a partially active dimeric intermediate (i.e., N2 left and right arrow I2). This is followed by a protein concentration dependent step in which I2 dissociates into compact inactive monomers (M) displaying enhanced hydrophobicity. The third unfolding transition, which is protein concentration independent, involves the complete unfolding of the monomeric state. Increasing NaCl concentrations destabilize N2 and appear to shift the equilibrium toward I2 whereas the stability of the monomeric intermediate M is enhanced. The binding of substrate or product analogue (i.e., glutathione or S-hexylglutathione) to the protein's active site stabilizes the native dimeric state (N2), causing the first two unfolding transitions to shift toward higher urea concentrations. The stability of M was not affected. The data implicate a region at/near the active site in domain I (most likely alpha-helix 2) as being highly unstable/flexible which undergoes local unfolding, resulting initially in I2 formation followed by a disruption in quaternary structure to a monomeric intermediate. The unfolding/refolding pathway is compared with those observed for other cytosolic GSTs and discussed in light of the different structural features at the subunit interfaces, as well as the evolutionary selection of this GST as a lens crystallin.     

Characterization and screening for mutations of the growth arrest-specific 11 (GAS11) and C16orf3 genes at 16q24.3 in breast cancer

BACKGROUND: Both fibroblast-mediated cytokine gene therapy and bone marrow transplantation (BMT) have proven to be efficient protocols for the recovery of bone marrow depression. In this report, the effects of fibroblast-mediated interleukin (IL)-6 gene therapy, in combination with BMT, on the recovery of irradiation-induced bone marrow depression were investigated. METHODS: NIH3T3 fibroblast cells engineered to secrete IL-6 (NIH3T3-IL-6) or NIH3T3 cells transduced with the neomycin gene (NIH3T3-Neo), in combination with 10(7), 10(6), or 10(5) syngeneic bone marrow cells, were implanted into irradiated mice. RESULTS: The platelets and white blood cells in the peripheral blood of the irradiated mice increased greatly 12 days after implantation of NIH3T3-IL-6 cells and BMT, the white blood cell counts were restored to a normal level 32 days after the combined therapy, and the platelet number was obviously higher than that in mice implanted with NIH3T3-Neo and BMT. Twenty and 25 days after the combined therapy, the mice showed accelerated recovery of colony-forming unit (CFU)-granulocyte/macrophages and CFU-megakaryocytes when compared with the mice implanted with NIH3T3-Neo cells and BMT. Ten days after lethal irradiation with gamma rays, the spleens formed more CFU-spleen in mice implanted with NIH3T3-IL-6 cells and BMT than in mice injected with phosphate-buffered saline or NIH3T3-Neo cells. Combined therapy with NIH3T3-IL-6 cell implantation and BMT delayed the survival period of the hematopoietic-depressed mice significantly when compared with therapy with NIH3T3-Neo cell implantation and BMT. CONCLUSIONS: These data demonstrated that the combined therapy of fibroblast-mediated IL-6 gene therapy and BMT could significantly promote the recovery of irradiation-induced hematopoietic depression.     

Curing characteristics of a composite matrix

The account describes investigations into the variations of specific volume and viscosity of a typical high-performance epoxy resin during various time-temperature cycles. The work was undertaken primarily to provide additional background information relating to the nature and causes of fibre kinking which has been observed in laminates comprising epoxy-carbon composite. The investigation has quantified certain characteristics of the resin which are presumed to be major contributory factors in the occurrence of fibre kinking. It is concluded that a simple solution to the general problem cannot be identified, although some possibilities are discussed for individual cases.     

NMR and sedimentation studies of a polymeric steric stabilizer for alumina

The sedimentation behavior of alumina powder has been studied in the presence of poly-vinylpyrrolidone (PVP) and poly(vinylpyrrolidone-co-vinyl acetate) (PVP/VA) in both thermodynamically “good” and “poor” solvents for the PVP homopolymer. PVP/VA provides higher sediment densities than does its PVP homopolymer counterpart. Solutionstate ¹³C-T₁ spin-lattice relaxation measurements were made on analogous mixtures both with and without alumina powder. The NMR results suggest that the PVP/VA copolymer is anchored to the alumina powder surface by means of VA moieties, whereas the PVP moieties extend into the continuous phase of the slurry medium. Thus, the higher settling densities that are observed in the presence of PVP/VA can be attributed to a steric stabilization mechanism. © 1993 John Wiley & Sons, Inc.     

The mechanism of discharge of sintered plate cadmium electrodes

Sintered plate Cd electrodes have been studied in alkaline solutions using potentiostatic and galvanostatic techniques. The behaviour is, in many ways, similar to that of a flat Cd electrode. A pseudo steady-state current is found due to the dissolution of Cd as Cd(OH)²–₄. At more anodic potentials passivation occurs due to the solid state formation of Cd(OH)₂. This model can account for the results obtained on galvanostatic discharge.     

A non‐isothermal finite element model for injection stretch‐blow molding of PET bottles with parametric studies

A non‐isothermal finite element (FE) model for the injection stretch‐blow molding (ISBM) process of polyethylene terephthalate (PET) bottles is presented in this paper. The constitutive behavior of PET is modeled by the physically based Buckley glass‐rubber model in form of UMAT in ABAQUS. The heat transfer between the stretch rod, the preform, and the mold is modeled. Particular attention is paid to thermal and contact modeling, material model, and selection of proper element types. Extensive FE simulations are carried out to model ISBM of a 20 g‐330 ml bottle made in plant tests. Comparisons of numerical results with the measurements demonstrate that the model can satisfactorily predict the bottle thickness and material distributions. Significant nonlinear differentials are found in strain, temperature, and temperature reduction rate in both bottle thickness and length direction during the process. A volume approach is therefore necessary for accurate predictions of final bottle properties because they are governed by orientation and crystallinity, which are highly temperature and strain dependent. Parametric studies on contact modeling and heat transfer coefficient are also conducted and the results are discussed. Polym. Eng. Sci. 44:1379–1390, 2004. © 2004 Society of Plastics Engineers.     

The effect of handle friction and inward or outward torque on maximum axial push force

OBJECTIVE: To investigate the relationship among friction, applied torque, and axial push force on cylindrical handles. BACKGROUND: We have earlier demonstrated that participants can exert greater contact force and torque in an "inward" movement of the hand about the long axis of a gripped cylinder (wrist flexion/forearm supination) than they can in an "outward" hand movement. METHOD: Twelve healthy participants exerted anteriorly directed maximum push forces along the long axis of aluminum and rubber handles while applying deliberate inward or outward torques, no torque (straight), and an unspecified (preferred) torque. RESULTS: Axial push force was 12% greater for the rubber handle than for the aluminum handle. Participants exerted mean torques of 1.1, 0.3, 2.5, and -2.0 Nm and axial push forces of 94, 85, 75, and 65 N for the preferred, straight, inward, and outward trials, respectively. Left to decide for themselves, participants tended to apply inward torques, which were associated with increased axial push forces. CONCLUSION: Axial push force was limited by hand-handle coupling--not the whole body's push strength. Participants appeared to intuitively know that the application of an inward torque would improve their maximum axial push force. Axial push forces were least when a deliberate torque was requested, probably because high levels of torque exertions interfered with the push. APPLICATION: A low-friction handle decreases maximum axial push force. It should be anticipated that people will apply inward torque during maximum axial push.     

Tandem mass spectrometry and nuclear magnetic resonance spectroscopy studies of Candida bombicola sophorolipids and product formed on hydrolysis by cutinase

Natural mixtures of sophorolipids produced by the yeast Candida bombicola have been analyzed by fast atom bombardment (FAB)-MS and collision-induced dissociation (CID)-MS. Some pure components have been analysed by two-dimensional NMR spectroscopy. The presence of acidic, lactonic, and O-acetylated forms and the position of double bonds in the fatty acid part of these glycolipids can be easily inferred from positive and negative ion FAB-mass spectra. Details about position of O-acetylation can be obtained from CID mass spectra of [M+H]+ and [M-H]- ions and from the NMR spectra. Differences in CID fragmentation between protonated and sodiated molecular ions are discussed in detail. Enzymatic hydrolysis of 6',6"-di-O-acetyl sophorolipid lactone by cutinase from Fusarium solani results specifically in the removal of the 6'-O-acetyl group, whereas the 6"-O-acetyl and lactone group are resistant. This specificity is explained from a three-dimensional model of the sophorolipid generated on the basis of the short 1H,1H distances as inferred from the NMR (ROESY) spectra.