Sequential action of ATP-dependent subunit conformational change and interaction between helical protrusions in the closure of the built-in lid of group II chaperonins

ATP drives the conformational change of the group II chaperonin from the open lid substrate-binding conformation to the closed lid conformation to encapsulate an unfolded protein in the central cavity. The detailed mechanism of this conformational change remains unknown. To elucidate the intra-ring cooperative action of subunits for the conformational change, we constructed Thermococcus chaperonin complexes containing mutant subunits in an ordered manner and examined their folding and conformational change abilities. Chaperonin complexes containing wild-type subunits and mutant subunits with impaired ATP-dependent conformational change ability or ATP hydrolysis activity, one by one, exhibited high protein refolding ability. The effects of the mutant subunits correlate with the number and order in the ring. In contrast, the use of a mutant lacking helical protrusion severely affected the function. Interestingly, these mutant chaperonin complexes also exhibited ATP-dependent conformational changes as demonstrated by small angle x-ray scattering, protease digestion, and changes in fluorescence of the fluorophore attached to the tip of the helical protrusion. However, their conformational change is likely to be transient. They captured denatured proteins even in the presence of ATP, whereas addition of ATP impaired the ability of the wild-type chaperonin to protect citrate synthase from thermal aggregation. These results suggest that ATP binding/hydrolysis causes the independent conformational change of the subunit, and further conformational change for the complete closure of the lid is induced and stabilized by the interaction between helical protrusions.     

Coffee pulp koji of Aspergillus sojae as stable immobilized catalyst of chlorogenate hydrolase

Chlorogenate hydrolase (EC 3.1.1.42, CHase) was highly induced in mycelia of Aspergillus sojae AKU 3312 grown in Czapek medium containing either instant coffee powder or coffee pulp as inducer. No CHase formation was observed in the mycelia when cultivated without the inducer. CHase was purified readily from CHase-induced mycelia to high homogeneity, and the purified CHase revealed the molecular weight of 180,000 consisting of two identical subunits of 88 kDa. Equimolar quinate (QA) and caffeate (CA) were confirmed on hydrolysis of chlorogenate (CGA). The purified CHase was only useful for a laboratory scale hydrolysis of CGA. For practical QA and CA production using scaled up hydrolysis of vegetable extracts of natural CGA resources, the enzyme activity of purified CHase decreased and denatured irreversibly. Preparation of coffee pulp koji and its application to QA and CA production were proposed instead of purified CHase. When coffee pulp koji was heated at 60°C for 30 min, CHase survived without any appreciable loss of enzyme activity while vegetative mycelial growth and spore germination were terminated. The heated coffee pulp koji thus prepared was effective itself as stable immobilized catalyst of CHase for QA and CA production from vegetable CGA resources such as coffee powders, coffee pulp, and others.     

Intracellular delivery of glutathione S-transferase-fused proteins into mammalian cells by polyethylenimine-glutathione conjugates

The glutathione S-transferase (GST)-fused protein expression system has been extensively used to generate a large quantity of proteins and has served for functional analysis in vitro. In this study, we developed a novel approach for the efficient intracellular delivery of GST-fused proteins into living cells to expand their usefulness up to in vivo use. Since protein cationization techniques are powerful strategies for efficient intracellular uptake by adsorptive-mediated endocytosis, GST-fused proteins were cationized by forming a complex with a polycationic polyethylenimine (PEI)-glutathione conjugate. On screening of protein transduction, optimized PEI-glutathione conjugate for protein transduction was characterized by a partly oligomerized mixture of PEI with average molecular masses of 600 (PEI600) modified with multiple glutathiones, which could have sufficient avidity for GST. Furthermore, enhanced endosomal escape of transduced GST-fused proteins was observed when they were delivered with a glutathione-conjugated PEI600 derivative possessing a hydroxybutenyl moiety. These results were confirmed by both intracellular confocal imaging of GST-fused green fluorescent protein and activation of an endogenous growth signal transduction pathway by a GST-fused constitutively active mutant of a kinase protein. These PEI-glutathione conjugates seem to be convenient molecular tools for protein transduction of widely used GST-fused proteins.     

Distinct physiological roles of two membrane-bound dehydrogenases responsible for D-sorbitol oxidation in Gluconobacter frateurii

Two different membrane-bound enzymes oxidizing D-sorbitol are found in Gluconobacter frateurii THD32: pyroloquinoline quinone-dependent glycerol dehydrogenase (PQQ-GLDH) and FAD-dependent D-sorbitol dehydrogenase (FAD-SLDH). In this study, FAD-SLDH appeared to be induced by L-sorbose. A mutant defective in both enzymes grew as well as the wild-type strain did, indicating that both enzymes are dispensable for growth on D-sorbitol. The strain defective in PQQ-GLDH exhibited delayed L-sorbose production, and lower accumulation of it, corresponding to decreased oxidase activity for D-sorbitol in spite of high D-sorbitol dehydrogenase activity, was observed. In the mutant strain defective in PQQ-GLDH, oxidase activity with D-sorbitol was much more resistant to cyanide, and the H(+)/O ratio was lower than in either the wild-type strain or the mutant strain defective in FAD-SLDH. These results suggest that PQQ-GLDH connects efficiently to cytochrome bo(3) terminal oxidase and that it plays a major role in L-sorbose production. On the other hand, FAD-SLDH linked preferably to the cyanide-insensitive terminal oxidase, CIO.     

Spatial variations in xylem sap flux density in evergreen oak trees with radial-porous wood: comparisons with anatomical observations

To estimate whole-tree water use when employing sap flow measurements, integration of the sap flux density (F _d) over the sapwood area is needed. Accordingly, it is necessary to obtain information on the characteristics of stem water transportation such as spatial variations in F _d and the active xylem area in the stem cross-section. Although evergreen oak trees with radial-porous wood represent a major component of secondary forests in western Japan, detailed information on their stem water transportation characteristics remains unclear. In the present study, we used the heat dissipation method (Granier method) to conduct measurements of azimuthal and radial variations in the F _d of Quercus glauca Thunb. ex Murray, a representative evergreen broad-leaved tree in western Japan. Further, by analyzing the anatomy of the xylem structure, we examined why F _d varies spatially in the stem cross-section. By using a dye solution injected into a radial hole bored into the tree trunk, we confirmed that the entire stem is hydroactive. We also compared the spatial variations in F _d and water conductivity per xylem area (K _s) which were estimated by using the observed vessel diameters and their density over the stem cross-section and Hagen–Poiseuille’s law. Azimuthal and radial variations in F _d reached about 60 and 50% of the maximum values, respectively, and could be explained by spatial variation in K _s. As a result, we obtained statistical parameters describing the spatial variation in F _d in Q. glauca and determined that whole-tree water use estimated from measurements in one direction had at most ±20% potential errors for studied trees.     

LET and ion-species dependence for mutation induction and mutation spectrum on hprt locus in normal human fibroblasts.

We have been studying LET and ion species dependence of RBE in mutation frequency and mutation spectrum of deletion pattern of exons in hprt locus. Normal human skin fibroblasts were irradiated with heavy-ion beams, such as carbon- (290 MeV/u and 135 MeV/u), neon- (230 MeV/u and 400 MeV/u), silicon- (490 MeV/u) and iron- (500 MeV/u) ion beams, generated by Heavy Ion Medical Accelerator in Chiba (HIMAC) at national Institute of Radiological Sciences (NIRS). Mutation induction in hprt locus was detected to measure 6-thioguanine resistant colonies and deletion spectrum of exons was analyzed by multiplex PCR. The LET-RBE curves of mutation induction for carbon- and neon-ion beams showed a peak around 75 keV/micrometers and 155 keV/micrometers, respectively. On the other hand, there observed no clear peak for silicon-ion beams. The deletion spectrum of exons was different in induced mutants among different ion species. These results suggested that quantitative and qualitative difference in mutation occurred when using different ion species even if similar LET values.     

Overexpression of poly(ADP-ribose) polymerase disrupts organization of cytoskeletal F-actin and tissue polarity in Drosophila.

Poly(ADP-ribose) polymerase (PARP) may play important roles in nuclear events such as cell cycle, cell proliferation, and maintenance of chromosomal stability. However, the exact biological role played by PARP or how PARP is involved in these cellular functions is still unclear. To elucidate the biological functions of PARP in vivo, we have constructed transgenic flies that overexpress Drosophila PARP in the developing eye primordia. These flies showed mild roughening of the normally smooth ommatidial lattice and tissue polarity disruption caused by improper rotation and chirality of the ommatidia. To clarify how this phenotypical change was induced, here we analyzed transgenic flies overexpressing PARP in the developing eye, embryo, and adult in detail. PARP mRNA level and the phenotype were enhanced in flies carrying more copies of the transgene. Developing eyes from third instar larvae were analyzed by using the neural cell marker to examine the involvement of PARP in cell fate. Morphological disorder of non-neuronal accessory cells was observed in PARP transgenic flies. Interestingly, overexpression of PARP did not interfere with the cell cycle or apoptosis, but it did disrupt the organization of cytoskeletal F-actin, resulting in aberrant cell and tissue morphology. Furthermore, heat-induced PARP expression disrupted organization of cytoskeletal F-actin in embryos and tissue polarity in adult flies. Because these phenotypes closely resembled mutants or transgenic flies of the tissue polarity genes, genetic interaction of PARP with known tissue polarity genes was examined. Transgenic flies expressing either PARP or RhoA GTPase in the eye were crossed, and co-expression of PARP suppressed the effect of RhoA GTPase. Our results indicate that PARP may play a role in cytoskeletal or cytoplasmic events in developmental processes of Drosophila.     

Vectorial redox reactions of physiological quinones. II. A study of transient semiquinone formation

Transient absorption changes during reduction of quinone in liposomes by external dithionite, in the absence and presence of initially trapped ferricyanide, were matched with absorption spectra of semiquinone and quinone in the blue region. Plastoquinone, ubiquinone-9 and phylloquinone, each having an isoprenoid side chain were compared with trimethyl-p-benzoquinone, ubiquinone-9 and menadione, which lack a long side chain.Semiquinone transients could only be observed by our spectroscopic technique during reduction of quinones lacking the chain. If Triton X-100 was added to the liposomes preparation semiquinone transients were also observed with the isoprenoid quinones. This result is consistent with the view that isoprenoid quinones build domains in the membranes, in which the life time of the semiquinone might be decreased by fast disproportionation, and to which dithionite has limited access.     

Predatory risk increased due to egg‐brooding in Armadillidium vulgare (Isopoda: Oniscidea)

Cost of reproduction is associated with a reduction in subsequent survival or future breeding success. A decrease in survival rate of parents during or after reproduction reduces the probability of their future reproduction. However, few studies have demonstrated such survival costs to parents. Females of Armadillidium vulgare hold their eggs in a marsupium and brood these until the young hatch. Caring for eggs in a marsupium seems to place a large burden on brooding females, and it restricts their predator avoidance behaviour. As such, costs of care may increase the mortality rates of brooding females. To reveal the costs of parental care, we examined the effects of egg brooding on behaviour and predation risk. Egg‐brooding females decreased speed of locomotion and rolling duration, and were killed by predators at a higher rate. Our results indicate that egg brooding in A. vulgare has costs in the form of predation risk.     

Effects of irradiated medium on chromatid aberrations in mammalian cells using double mylar dishes.

We examined the potential contribution of irradiated medium on the bystander effect using custom made double-mylar stainless steel rings. Exponentially growing Human-hamster hybrid (AL) cells were plated on either one or both sides of double-mylar dishes 2-4 days before irradiation. One side (with or without cells) was irradiated with alpha particles using the track segment mode of a 4 MeV Van de Graaff accelerator at the Radiological Research Accelerator Facilities of Columbia University. Since alpha particles can only traverse a very limited distance, cells plated on the other side of a medium-filled mylar dish will not be irradiated by the alpha particles. The results of chromosomal aberrations on un-irradiated target cells that were attached to the top mylar layer indicate that the number of chromatid-type aberrations was higher when there was a bottom layer of cells in the medium filled chambers than just medium alone. Furthermore, when transferring the medium from these cell-irradiated dishes to fresh AL cultures, chromatid-type aberrations were produced in the un-irradiated fresh cells. In contrast, medium irradiated in the absence of cells had no effect on chromatid aberrations. These results suggest that certain modulating factors secreted from the irradiated cells on the bottom mylar layer into the medium, induce chromatin damage in the un-irradiated, bystander cells.