Occurrence of fructosyl-amino acid oxidase-reactive compounds in fungal cells

Fructosyl-amino acid oxidase (FAOD)-reactive fraction (FRY) was found in commercial yeast extract. FRY showed very hydrophilic property and was adsorbed to phenylboronate silica gel, indicating that it contained the Amadori compound. TLC and amino acid analyses revealed that glucosone, lysine, and arginine were produced from FRY after incubation with FAOD. TOF-MS analysis confirmed that FRY is a mixture of fructosyl lysine and fructosyl arginine. These compounds were also detected in mycelial extract of an FAOD-producer, Aspergillus terreus GP1, grown on the minimum medium, suggesting that a glycation reaction occurs in fungal cells and that FAOD acts toward the resultant Amadori compounds.     

Biotin-labeled abscisic acid as a probe for investigating abscisic acid binding sites on plasma membranes of barley aleurone protoplasts

Plant hormone abscisic acid (ABA) plays important roles in dormancy and stress responses, but its binding sites have not yet been fully elucidated. In this report, we suggest the utility of biotin-labeled abscisic acid (bioABA) as a probe to investigate ABA-binding sites on the plasma membrane of barley aleurone protoplasts. BioABA was approximately 100 times less effective than ABA in inhibiting expression of gibberellin-inducible alpha-amylase and in inducing expression of a reporter gene fused to the dehydrin promoter. To ascertain that bioABA could bind to ABA-binding sites on the plasma membrane, we used fluorescence flow cytometry to measure the fluorescence intensity of aleurone protoplasts treated with a combination of bioABA and fluorescence-labeled streptavidin. Addition of bioABA increased the fluorescence of aleurone protoplasts in a concentration-dependent manner, but addition of non-active bioABA derivatives did not. Furthermore, the increase in fluorescence intensity observed upon addition of bioABA was eliminated by co-treatment with excess ABA, but it was not eliminated by co-treatment with other plant hormones. These results suggest that bioABA binds to ABA-binding sites, and that bioABA should be a valuable probe for investigating ABA-binding sites on the plasma membrane.     

13-Deoxytedanolide, a marine sponge-derived antitumor macrolide, binds to the 60S large ribosomal subunit

13-Deoxytedanolide is a potent antitumor macrolide isolated from the marine sponge Mycale adhaerens. In spite of its remarkable activity, the mode of action of 13-deoxytedanolide has not been elucidated. [11-3H]-(11S)-13-Deoxydihydrotedanolide derived from the macrolide was used for identifying the target molecule from the yeast cell lysate. Fractionation of the binding protein revealed that the labeled 13-deoxytedanolide derivative strongly bound to the 80S ribosome as well as to the 60S large subunit, but not to the 40S small subunit. In agreement with this observation, 13-deoxytedanolide efficiently inhibited the polypeptide elongation. Interestingly, competition studies demonstrated that 13-deoxytedanolide shared the binding site on the 60S large subunit with pederin and its marine-derived analogues. These results indicate that 13-deoxytedanolide is a potent protein synthesis inhibitor and is the first macrolide to inhibit the eukaryotic ribosome.     

RNA interference during spermatogenesis in mice

Spermatogenesis consists of complex cellular and developmental processes, such as the mitotic proliferation of spermatogonial stem cells, meiotic division of spermatocytes, and morphogenesis of haploid spermatids. In this study, we show that RNA interference (RNAi) functions throughout spermatogenesis in mice. We first carried out in vivo DNA electroporation of the testis during the first wave of spermatogenesis to enable foreign gene expression in spermatogenic cells at different stages of differentiation. Using prepubertal testes at different ages and differentiation stage-specific promoters, reporter gene expression was predominantly observed in spermatogonia, spermatocytes, and round spermatids. This method was next applied to introduce DNA vectors that express small hairpin RNAs, and the sequence-specific reduction in the reporter gene products was confirmed at each stage of spermatogenesis. RNAi against endogenous Dmc1, which encodes a DNA recombinase that is expressed and functionally required in spermatocytes, led to the same phenotypes observed in null mutant mice. Thus, RNAi is effective in male germ cells during mitosis and meiosis as well as in haploid cells. This experimental system provides a novel tool for the rapid, first-pass assessment of the physiological functions of spermatogenic genes in vivo.     

One rotary mechanism for F1-ATPase over ATP concentrations from millimolar down to nanomolar

F₁-ATPase is a rotary molecular motor in which the central γ-subunit rotates inside a cylinder made of α₃β₃-subunits. The rotation is driven by ATP hydrolysis in three catalytic sites on the β-subunits. How many of the three catalytic sites are filled with a nucleotide during the course of rotation is an important yet unsettled question. Here we inquire whether F₁ rotates at extremely low ATP concentrations where the site occupancy is expected to be low. We observed under an optical microscope rotation of individual F₁ molecules that carried a bead duplex on the γ-subunit. Time-averaged rotation rate was proportional to the ATP concentration down to 200 pM, giving an apparent rate constant for ATP binding of 2 × 10⁷ M⁻¹s⁻¹. A similar rate constant characterized bulk ATP hydrolysis in solution, which obeyed a simple Michaelis-Menten scheme between 6 mM and 60 nM ATP. F₁ produced the same torque of ~40 pN·nm at 2 mM, 60 nM, and 2 nM ATP. These results point to one rotary mechanism governing the entire range of nanomolar to millimolar ATP, although a switchover between two mechanisms cannot be dismissed. Below 1 nM ATP, we observed less regular rotations, indicative of the appearance of another reaction scheme.     

Autoregulation and homodimerization are involved in the activation of the plant steroid receptor BRI1

The leucine-rich-repeat receptor serine/threonine kinase, BRI1, is a cell-surface receptor for brassinosteroids (BRs), the steroid hormones of plants, yet its activation mechanism is unknown. Here, we report a unique autoregulatory mechanism of BRI1 activation. Removal of BRI1's C terminus leads to a hypersensitive receptor, indicated by suppression of dwarfism of BR-deficient and BR-perception mutants and by enhanced BR signaling as a result of elevated phosphorylation of BRI1. Several sites in the C-terminal region can be phosphorylated in vitro, and transgenic Arabidopsis expressing BRI1 mutated at these sites demonstrates an essential role of phosphorylation in BRI1 activation. BRI1 is a ligand-independent homo-oligomer, as evidenced by the transphosphorylation of BRI1 kinase in vitro, the dominant-negative effect of a kinase-inactive BRI1 in transgenic Arabidopsis, and coimmunoprecipitation experiments. Our results support a BRI1-activation model that involves inhibition of kinase activity by its C-terminal domain, which is relieved upon ligand binding to the extracellular domain.     

Protein expressed by the ho2 gene of the cyanobacterium Synechocystis sp. PCC 6803 is a true heme oxygenase. Properties of the heme and enzyme complex

Two isoforms of a heme oxygenase gene, ho1 and ho2, with 51% identity in amino acid sequence have been identified in the cyanobacterium Synechocystis sp. PCC 6803. Isoform-1, Syn HO-1, has been characterized, while isoform-2, Syn HO-2, has not. In this study, a full-length ho2 gene was cloned using synthetic DNA and Syn HO-2 was demonstrated to be highly expressed in Escherichia coli as a soluble, catalytically active protein. Like Syn HO-1, the purified Syn HO-2 bound hemin stoichiometrically to form a heme-enzyme complex and degraded heme to biliverdin IXalpha, CO and iron in the presence of reducing systems such as NADPH/ferredoxin reductase/ferredoxin and sodium ascorbate. The activity of Syn HO-2 was found to be comparable to that of Syn HO-1 by measuring the amount of bilirubin formed. In the reaction with hydrogen peroxide, Syn HO-2 converted heme to verdoheme. This shows that during the conversion of hemin to alpha-meso-hydroxyhemin, hydroperoxo species is the activated oxygen species as in other heme oxygenase reactions. The absorption spectrum of the hemin-Syn HO-2 complex at neutral pH showed a Soret band at 412 nm and two peaks at 540 nm and 575 nm, features observed in the hemin-Syn HO-1 complex at alkaline pH, suggesting that the major species of iron(III) heme iron at neutral pH is a hexa-coordinate low spin species. Electron paramagnetic resonance (EPR) revealed that the iron(III) complex was in dynamic equilibrium between low spin and high spin states, which might be caused by the hydrogen bonding interaction between the distal water ligand and distal helix components. These observations suggest that the structure of the heme pocket of the Syn HO-2 is different from that of Syn HO-1.     

Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins

By combining translation and membrane integration/translocation systems, we have constructed a novel cell-free system for the production of presecretory and integral membrane proteins in vitro. A totally defined, cell-free system reconstituted from a minimal number of translation factors was supplemented with urea-washed inverted membrane vesicles (U-INVs) prepared from Escherichia coli, as well as with purified proteins mediating membrane targeting of presecretory and integral membrane proteins. Initially, efficient membrane translocation of a presecretory protein (pOmpA) was obtained simply by the addition of only SecA and SecB. Proteinase K digestion clearly showed the successful translocation of pOmpA inside the vesicles. Next, integration of an inner membrane protein (MtlA) into U-INVs was achieved in the presence of only SRP (Ffh) and SR (FtsY). Finally, a membrane protein possessing a large periplasmic region (FtsQ) and therefore requiring both factors (SRP/SR and SecA/SecB) for membrane integration/translocation was also shown to be integrated correctly in this cell-free system. Thus, our novel cell-free system provides not only an efficient strategy for the production of membrane-related proteins but also an improved platform for the biological study of protein translocation and integration mechanisms.