Characterization of archaeal group II chaperonin-ADP-metal fluoride complexes: implications that group II chaperonins operate as a "two-stroke engine"

Group II chaperonins, found in Archaea and in the eukaryotic cytosol, act independently of a cofactor corresponding to GroES of group I chaperonins. Instead, the helical protrusion at the tip of the apical domain forms a built-in lid of the central cavity. Although many studies on the lid's conformation have been carried out, the conformation in each step of the ATPase cycle remains obscure. To clarify this issue, we examined the effects of ADP-aluminum fluoride (AlFx) and ADP-beryllium fluoride (BeFx) complexes on alpha-chaperonin from the hyperthermophilic archaeum, Thermococcus sp. strain KS-1. Biochemical assays, electron microscopic observations, and small angle x-ray scattering measurements demonstrate that alpha-chaperonin incubated with ADP and BeFx exists in an asymmetric conformation; one ring is open, and the other is closed. The result indicates that alpha-chaperonin also shares the inherent functional asymmetry of bacterial and eukaryotic cytosolic chaperonins. Most interestingly, addition of ADP and BeFx induced alpha-chaperonin to encapsulate unfolded proteins in the closed ring but did not trigger their folding. Moreover, alpha-chaperonin incubated with ATP and AlFx or BeFx adopted a symmetric closed conformation, and its functional turnover was inhibited. These forms are supposed to be intermediates during the reaction cycle of group II chaperonins.     

Spindle checkpoint signaling requires the mis6 kinetochore subcomplex, which interacts with mad2 and mitotic spindles

The spindle checkpoint coordinates cell cycle progression and chromosome segregation by inhibiting anaphase promoting complex/cyclosome until all kinetochores interact with the spindle properly. During early mitosis, the spindle checkpoint proteins, such as Mad2 and Bub1, accumulate at kinetochores that do not associate with the spindle. Here, we assess the requirement of various kinetochore components for the accumulation of Mad2 and Bub1 on the kinetochore in fission yeast and show that the necessity of the Mis6-complex and the Nuf2-complex is an evolutionarily conserved feature in the loading of Mad2 onto the kinetochore. Furthermore, we demonstrated that Nuf2 is required for maintaining the Mis6-complex on the kinetochore during mitosis. The Mis6-complex physically interacts with Mad2 under the condition that the Mad2-dependent checkpoint is activated. Ectopically expressed N-terminal fragments of Mis6 localize along the mitotic spindle, highlighting the potential binding ability of Mis6 not only to the centromeric chromatin but also to the spindle microtubules. We propose that the Mis6-complex, in collaboration with the Nuf2-complex, monitors the spindle-kinetochore attachment state and acts as a platform for Mad2 to accumulate at unattached kinetochores.     

Quinolactacide, a new quinolone insecticide from Penicillium citrinum Thom F 1539

By a screening program searching for new pesticides from fungal sources, an insecticidal compound was isolated from Penicillium citrinum F 1539. The compound, named quinolactacide, was novel and showed 88% mortality against green peach aphids (Myzus persicae) at 250 ppm. Its structure was determined by spectroscopic techniques.     

Costal-2: a scaffold for kinases mediates Hedgehog signaling

Drosophila genetics has identified several components of the Hedgehog signaling pathway, but the mechanism by which they act remains elusive. In this issue of Developmental Cell, a report by Zhang et al. provides evidence that the kinesin-related protein Costal-2 forms a multi-component scaffold that mediates Hedgehog signaling.     

Effects of AH-9700, (+)-pentazocine, DTG and oxybutynin on micturition in anesthetized rats with acetone-induced cystitis

We investigated the effects of AH-9700 (1-[2-(3,4-dihydro-6,7-dimethyl-2-naphthalenyl)ethyl] pyrrolidine fumarate; a novel sigma receptor ligand), (+)-pentazocine and 1,3-di-o-tolylguanidine (DTG) (two typical sigma receptor ligands), and oxybutynin (a currently used anti-pollakiuria drug) on cystometrograms in anesthetized rats with 30% acetone-induced cystitis. Compared to sham-treated rats, acetone-treated cystitis models exhibited an increase in urinary frequency during continuous filling cystometry. Intravenous administration of AH-9700 (1-5 mg/kg), (+)-pentazocine or DTG to the rats with cystitis dose-dependently prolonged micturition intervals and increased the micturition threshold pressure. Oxybutynin (1 mg/kg. i.v.) also extended micturition intervals, but decreased the micturition pressure. These results indicate that AH-9700, (+)-pentazocine and DTG improve abnormal frequent urination caused by acetone-induced cystitis in a manner different from that of oxybutynin.     

Role of mitochondria in alcoholic liver injury

Oxidative stress and oxygen-derived free radicals are well known to play an important role in the pathogenesis of ethanol-associated liver injury. Active oxidants produced during ethanol metabolism induce mitochondrial membrane depolarization and permeability changes in cultured hepatocytes. These mitochondrial alterations (loss of DeltaPsim and mitochondrial permeability transition [MPT]) are now recognized as a key step in apoptosis. In recent studies, including ours, the MPT has been identified as a key step for the induction of mitochondrial cytochrome c release and caspase activation by ethanol. In addition, chronic and/or acute ethanol modulates intracellular, especially mitochondrial, antioxidant levels, leading to the increased susceptibility to alcoholic liver injury induced by several apoptotic stimuli. In this review, we address the mechanism of mitochondrial alterations and liver injury induced by ethanol.     

Changes in activity coefficient gamma(w) of water and the foaming capacity of protein during hydrolysis

The changes in the interaction between food proteins and water and in their surface functional property during enzymatic hydrolysis were investigated. Ovalbumin, a soy protein isolate (SPI), and casein were hydrolyzed with trypsin, and the degree of hydrolysis, water activity a(w), and foaming capacity of each hydrolysate were measured. Ovalbumin showed the minimum value for a(w), and the values for SPI and casein progressively decreased during hydrolysis. Therefore, the activity coefficient of water, gamma(w) (=a(w)/x(w), where x(w) is the mole fraction of water) was obtained to remove the influence of mole change and to examine the interaction of protein hydrolysates with water. In order to calculate x(w) in a sample during protein hydrolysis, a method for roughly estimating the number of moles of the protein hydrolysate in a solution was developed. The strategy was to modify the TNBS (2,4,6-trinitrobenzenesulfonic acid) method and to combine this method with the modified Ellman method and the determination of lysine by an amino acid analyzer. During enzymatic hydrolysis, each protein sample showed a minimum gamma(w) value and maximum foaming capacity.     

Characterization of S-sulfokeratein from pig hair and its use as a modifier of type I collagen gel

S-sulfokeratein is prepared through S-sulfonation after the cleavage of disulfide bonds in keratin using ditiothreitol in urea. S-sulfokeratein is composed of two fractions, matrix and microfibril components, and S-sulfokeratein from the matrix component (Bs) can regenerate disulfide bonds. In this study, the effects of Bs and partially reduced Bs on type I collagen self-assembly and properties of reconstructed Bs- or partially reduced Bs-collagen gel were investigated. It was proved that collagen self-assembly was accelerated by the increased amount of added Bs, but partially reduced Bs with 10 mg DTT/100 mg Bs (Bs-10) did not affect the ratio of collagen self-assembly. The mechanical strength of Bs-collagen gel proved to be lower than control, but that of Bs-10-collagen gel was times higher than that of control.