Identification of <Emphasis Type="Italic">mokB</Emphasis> involved in monacolin K biosynthesis in <Emphasis Type="Italic">Monascus pilosus</Emphasis>

Monacolin K (MK), which is widely used as an antihypercholesterolemia medicine, is produced as a fungal secondary metabolite through the polyketide pathway. The MK biosynthetic gene cluster proposed for Monascus pilosus BCRC38072 was also identified in M. pilosus NBRC4480. The mokB gene, located at the end of the putative gene cluster and possibly encoding polyketide synthase, was disrupted. The mokB disruptant did not produce MK, but accumulated an intermediate that was confirmed to be monacolin J, indicating that mokB encodes the polyketide synthase responsible for the biosynthesis of side-chain diketide moiety.     

Enhancement of menadione stress tolerance in yeast by accumulation of hypotaurine and taurine: co-expression of cDNA clones, from Cyprinus carpio, for cysteine dioxygenase and cysteine sulfinate decarboxylase in Saccharomyces cerevisiae

Taurine is known to function as a protectant against various stresses in animal cells. In order to utilize taurine as a compatible solute for stress tolerance of yeast, isolation of cDNA clones for genes encoding enzymes involved in biosynthesis of taurine was attempted. Two types of cDNA clones corresponding to genes encoding cysteine dioxygenase (CDO1 and CDO2) and a cDNA clone for cysteine sulfinate decarboxylase (CSD) were isolated from Cyprinus carpio. Deduced amino acid sequences of the two CDOs and that of CSD showed high similarity to those of CDOs and those of CSDs from other organisms, respectively. The coding regions of CDO1, CDO2, and CSD were subcloned into an expression vector, pESC-TRP, for Saccharomyces cerevisiae. Furthermore, to enhance the efficiency of synthesis of taurine in S. cerevisiae, a CDO–CSD fusion was designed and expressed. Expression of CDO and CSD proteins, or the CDO–CSD fusion protein was confirmed by Western blot analysis. HPLC analysis showed that the expression of the proteins led to enhancement of the accumulation level of hypotaurine, a precursor of taurine, rather than taurine. The yeast cells expressing corresponding genes showed tolerance to oxidative stress induced by menadione, but not to freezing–thawing stress.     

The role of {beta}-TrCP1 and {beta}-TrCP2 in circadian rhythm generation by mediating degradation of clock protein PER2

The mammalian circadian clock proteins undergo a daily cycle of accumulation followed by phosphorylation and degradation. The mechanism by which clock proteins undergo degradation has not been fully understood. Circadian clock protein PERIOD2 (PER2) is shown to be the potential target of F-box protein beta-TrCP1, a component of ubiquitin E3 ligase. Here, we show that beta-TrCP2 as well as beta-TrCP1 target PER2 protein in vitro. We also identified beta-TrCP binding site (m2) of PER2 being recognized by both beta-TrCP1 and beta-TrCP2. Luciferase-PER2 fusion system revealed that m2 site was responsible for the stability of PER2. The role of beta-TrCP1 and beta-TrCP2 in circadian rhythm generation was analysed by real-time reporter assay revealing that siRNA-mediated suppressions of beta-TrCP1 and/or beta-TrCP2 attenuate circadian oscillations in NIH3T3 cell. beta-TrCP1-deficient mice, however, showed normal period length, light-induced phase-shift response in behaviour and normal expression of PER2, suggesting that beta-TrCP1 is dispensable for the central clock in the suprachiasmatic nucleus. Our study indicates that beta-TrCP1 and beta-TrCP2 were involved in the cell autonomous circadian rhythm generation in culture cells, although the role of beta-TrCP2 in the central clock in the suprachiasmatic nucleus remains to be elucidated.     

Aβ42 Mutants with Different Aggregation Profiles Induce Distinct Pathologies in Drosophila

Aggregation of the amyloid-β-42 (Aβ42) peptide in the brain parenchyma is a pathological hallmark of Alzheimer''s disease (AD), and the prevention of Aβ aggregation has been proposed as a therapeutic intervention in AD. However, recent reports indicate that Aβ can form several different prefibrillar and fibrillar aggregates and that each aggregate may confer different pathogenic effects, suggesting that manipulation of Aβ42 aggregation may not only quantitatively but also qualitatively modify brain pathology. Here, we compare the pathogenicity of human Aβ42 mutants with differing tendencies to aggregate. We examined the aggregation-prone, EOFAD-related Arctic mutation (Aβ42Arc) and an artificial mutation (Aβ42art) that is known to suppress aggregation and toxicity of Aβ42 in vitro. In the Drosophila brain, Aβ42Arc formed more oligomers and deposits than did wild type Aβ42, while Aβ42art formed fewer oligomers and deposits. The severity of locomotor dysfunction and premature death positively correlated with the aggregation tendencies of Aβ peptides. Surprisingly, however, Aβ42art caused earlier onset of memory defects than Aβ42. More remarkably, each Aβ induced qualitatively different pathologies. Aβ42Arc caused greater neuron loss than did Aβ42, while Aβ42art flies showed the strongest neurite degeneration. This pattern of degeneration coincides with the distribution of Thioflavin S-stained Aβ aggregates: Aβ42Arc formed large deposits in the cell body, Aβ42art accumulated preferentially in the neurites, while Aβ42 accumulated in both locations. Our results demonstrate that manipulation of the aggregation propensity of Aβ42 does not simply change the level of toxicity, but can also result in qualitative shifts in the pathology induced in vivo.     

Biosynthetic processing of cathepsins and lysosomal degradation are abolished in asparaginyl endopeptidase-deficient mice

Asparaginyl endopeptidase (AEP)/legumain, an asparagine-specific cysteine proteinase in animals, is an ortholog of plant vacuolar processing enzyme (VPE), which processes the exposed asparagine residues of various vacuolar proteins. In search for its physiological role in mammals, here we generated and characterized AEP-deficient mice. Although their body weights were significantly reduced, they were normally born and fertile. In the wild-type kidney where the expression of AEP was exceedingly high among various organs, the localization of AEP was mainly found in the lamp-2-positive late endosomes in the apical region of the proximal tubule cells. In these cells of AEP-deficient mice, the lamp-2-positive membrane structures were found to be greatly enlarged. These aberrant lysosomes, merged with the late endosomes, accumulated electron-dense and membranous materials. Furthermore, the processing of the lysosomal proteases, cathepsins B, H, and L, from the single-chain forms into the two-chain forms was completely defected in the deficient mice. Thus, the AEP deficiency caused the accumulation of macromolecules in the lysosomes, highlighting a pivotal role of AEP in the endosomal/lysosomal degradation system.     

A metabolic model describing the H2O2 elimination by mammalian cells including H2O2 permeation through cytoplasmic and peroxisomal membranes: comparison with experimental data

We have constructed a metabolic model describing the H2O2 elimination by mammalian cells. It comprises three compartments (medium, cytosol, and peroxisome) separated by cytoplasmic and peroxisomal membranes, and H2O2 moves across the membranes with different permeation rate constants. Catalase localizes to peroxisomes, while glutathione peroxidase (GPx) and GSH recycling system (glutathione reductase (GR) and the oxidative pentose phosphate pathway (PPP)) localize to cytosol. The rates of individual enzyme reactions were computed using the experimentally determined activities and rate equations known for mammalian enzymes. Using the model, the concentration dependence of H2O2 elimination rate was obtained by numerical simulation and was compared with experimental data obtained previously with cultured mammalian cells (fibroblasts, human umbilical vein endothelial cells (HUVEC), and PC12 cells). The model was shown to be able to reproduce the data well by assuming appropriate values for the permeability rate constants. The H2O2 permeability coefficients thus estimated for cytoplasmic and peroxisomal membranes were in the same order of magnitude, except that the value for cytoplasmic membrane of PC12 cell was significantly smaller. The results suggest that the membrane permeability is one of the rate-limiting factors in the H2O2 elimination by mammalian cells. Using the model and estimated parameter values, we have examined the rate-limiting enzyme of the metabolic system, as well as the intracellular H2O2 concentration under steady-state and non-steady-state conditions.     

Characterization of anisocotylous leaf formation in Streptocarpus wendlandii (Gesneriaceae): significance of plant growth regulators

BACKGROUND AND AIMS: Unifoliate species of Gesneriaceae are unique, as they bear only one leaf throughout their life history. The development of this leaf (termed a macrocotyledon) derived from one of two cotyledons is intriguing. The other cotyledon does not develop further and is termed a microcotyledon. This process of unequal cotyledon development is termed anisocotyly. In this study the process of macrocotyeldon formation was studied and the effects of plant hormones on the macrocotyledon development were investigated. METHODS: Streptocarpus wendlandii was chosen as the main subject material, as it was found to be suitable for experimental studies in laboratory conditions. Morphological analyses were carried out with light and scanning electron microscopy. Plant hormones were applied exogenously. KEY RESULTS: The macrocotyledon of S. wendlandii is produced through cell division activity in the basal meristem of the enlarging cotyledon. The newly developed region in the macrocotyledon displayed distinct morphological changes, including the formation of long, needle-shaped trichomes. The newly formed region was surrounded by lateral veins. No such change was observed in the microcotyledon. Furthermore, it was shown that development of anisocotyly is suppressed by the application of cytokinin, resulting in the formation of two nearly equal-sized cotyledons. Both cotyledons displayed macrocotyledon characteristics. This observation in S. wendlandii was confirmed using Monophyllaea glabra, another unifoliate species in the same family. CONCLUSIONS: It is proposed that developmental changes of the macrocotyledon have characteristics of a developmental phase-change, and cytokinins may be involved in its formation. These results are discussed in the light of current knowledge of phase-change transitions in plant vegetative development.     

Secreted frizzled-related protein-1 binds directly to Wingless and is a biphasic modulator of Wnt signaling

Secreted Frizzled-related protein-1 (sFRP-1) contains a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzleds. To facilitate the biochemical and biological analysis of sFRP-1, we developed a mammalian recombinant expression system that yields approximately 3 mg of purified protein/liter of conditioned medium. Using this recombinant protein, we demonstrated that sFRP-1 and Wg (wingless) interact in enzyme-linked immunosorbent and co-precipitation assays. Surprisingly, a derivative lacking the cysteine-rich domain retained the ability to bind Wg. Cross-linking experiments performed with radioiodinated sFRP-1 provided definitive evidence that sFRP-1 and Wg bind directly to each other. Besides detecting a cross-linked complex consistent in size with 1:1 stoichiometry of sFRP-1 and Wg, we also observed a larger complex whose size suggested the presence of a second sFRP-1 molecule. The formation of both complexes was markedly enhanced by an optimal concentration of exogenous heparin, emphasizing the potential importance of heparan-sulfate proteoglycan in Wnt binding and signaling. sFRP-1 exerted a biphasic effect on Wg activity in an armadillo stabilization assay, increasing armadillo level at low concentrations but reducing it at higher concentrations. These results provide new insights about the Wnt binding and biological activity of sFRPs.