Chestnut pellicle for the recovery of gold

Recovery of Au(III) from hydrochloric acid medium by using crosslinked chestnut pellicle (CCP) gel was studied. Strong selectivity was observed for Au(III) showing negligible affinity for other precious metals and some base metal ions tested. The adsorption isotherm study exhibited the maximum loading capacity of the gel as high as 10.6 mol or about 2.1 kg gold per kg dry weight of gel. The reduction of Au(III) ion to elemental form during adsorption process is expected to be the reason of high selectivity and high capacity for Au(III). Kinetic studies at various temperatures confirm an endothermic adsorption process following the pseudo-first order rate law.     

Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae

(E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter⁻¹) rather than GGOH (0.2 mg liter⁻¹) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter⁻¹ GGOH and 6.5 mg liter⁻¹ squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter⁻¹ GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering.(E,E,E)-Geranylgeraniol (GGOH) can be used as an important ingredient for perfumes and as a desirable raw material for synthesizing vitamins A and E (4, 13). It is also known to induce apoptosis in various cancer and tumor cell lines (24, 36). GGOH is the dephosphorylated derivative of (E,E,E)-geranylgeranyl diphosphate (GGPP) (Fig. ​(Fig.1).1). GGPP is a significant intermediate of ubiquinone and carotenoid biosyntheses, especially in carotenoid-producing microorganisms and plant cells. It is also utilized as the lipid anchor of geranylgeranylated proteins. In the yeast Saccharomyces cerevisiae, GGPP is synthesized by GGPP synthase (GGPS), encoded by the BTS1 gene, which catalyzes the condensation of farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) rather than the successive addition of IPP molecules to dimethylallyl diphosphate, geranyl diphosphate, and FPP that is detected in mammalian tissues (14). Biologically synthesized GGOH comprises only (E,E,E)-geometric isomers, and only the (E,E,E)-isomers have significant biological activities (23). The chemically synthesized form is usually obtained as mixtures of (E)- and (Z)-isomers and thus has lower potency. Therefore, there is a greater possibility of attaining efficient production of (E,E,E)-GGOH through fermentative production.Open in a separate windowFIG. 1.Biosynthetic pathway for GGOH in S. cerevisiae. The solid arrows indicate the one-step conversions in the biosynthesis, and the dashed arrows indicate the several steps. Intermediates: HMG-CoA, 3-hydroxy-3-methylflutaryl coenzyme A; DMAPP, dimethylallyl diphosphate. Enzymes: HMG-R, HMG-coenzyme A reductase (encoded by the HMG1 gene); FPS, FPP synthase (ERG20).Some yeast strains accumulate ergosterol up to 4.6% dry mass (1). Thus, yeasts have the potential to produce large amounts of GGOH if it is possible to enhance and redirect the metabolic flux to GGOH synthesis. The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-R), encoded by the HMG1 gene has been shown to be the major rate-limiting enzyme in the mevalonate pathway in S. cerevisiae (12). Overproduction of the catalytic domain of HMG-R in an S. cerevisiae strain resulted in squalene accumulation of up to 1% (27) and 2% (8) dry mass but did not cause any difference in the contents of isoprenoid alcohols such as farnesol (FOH) and geraniol (27). These results suggest that squalene is preferably accumulated rather than GGOH when the mevalonate pathway is enhanced by overexpression of the HMG1 gene. Squalene is synthesized through the condensation of two molecules of FPP catalyzed by squalene synthase (SQS) encoded by the ERG9 gene in S. cerevisiae (Fig. ​(Fig.1).1). The addition of an SQS inhibitor to cultures of S. cerevisiae strains resulted in the production of considerable amounts of FOH (∼77.5 mg liter⁻¹) and relatively small amounts of GGOH (∼2.2 mg liter⁻¹) (20). It has also been reported that SQS-deficient (Δerg9) S. cerevisiae strains, which are sterol auxotrophic, accumulated FPP in their cells (35) and excreted 1.3 mg liter⁻¹ of FOH into the culture medium (5). Therefore, inactivation of SQS seems to enhance FOH rather than GGOH production. This is probably because of the low GGPS activity in S. cerevisiae. Indeed, a carotenoid-producing Rhodotorula yeast strain showed higher GGOH (24.4 mg liter⁻¹) than FOH (4.4 mg liter⁻¹) production on cultivation with an SQS inhibitor (20). Our group previously found that GGOH production could be enhanced by overexpression of the BTS1 gene in S. cerevisiae without SQS inhibition. In addition, coexpression of a fusion of the BTS1 and farnesyl diphosphate synthetase (ERG20) genes along with the HMG1 gene resulted in the production of a substantial amount of GGOH with only a small amount of FOH (C. Ohto, M. Muramatsu, E. Sakuradani, S. Shimizu, and S. Obata, submitted for publication).These results suggest that GGOH can be produced from GGPP through some endogenous phosphatase activities when GGPP synthesis is enhanced. We therefore hypothesized that enhancement of the phosphatase activity could increase the productivity of GGOH. However, it is not clear what kind of phosphatase enhances the GGOH production. It has been reported that the products of the diacylglycerol diphosphate phosphatase (DPP1) gene and lipid phosphate phosphatase (LPP1) gene account for most of the FPP and GGPP phosphatase activities in a particulate (membrane associated) fraction of S. cerevisiae (9). In this study, we found that GGOH production could be enhanced by overexpression of these phosphatase genes. We also demonstrated that overexpression of the BTS1-DPP1 and BTS1-ERG20 fusion genes along with the HMG1 gene further increased GGOH production. Finally, we constructed a high-level GGOH-producing yeast available for industrial processes involving multicopy integration vectors. The productivity of GGOH was evaluated in test tube cultures and 10-liter jar fermentors.     

Weak gel of chitin with ionic liquid, 1-allyl-3-methylimidazolium bromide

This paper reports the formation of weak gel of chitin with an ionic liquid, 1-allyl-3-methylimidazolium bromide (IL). When a mixture of 5% (w/w) chitin with IL was heated at 100 °C for 48 h, the clear liquid was obtained. The experimental process was observed by the CCD camera view and the SEM analysis. From a mixture of chitin with IL in the higher concentration (7%, w/w), a more viscous material, i.e., a gel-like material was obtained. The rheological evaluations showed that both 5% (w/w) and 7% (w/w) chitins with IL behaved as weak gels.     

Production of geranylgeraniol on overexpression of a prenyl diphosphate synthase fusion gene in Saccharomyces cerevisiae

An acyclic diterpene alcohol, (E,E,E)-geranylgeraniol (GGOH), is one of the important compounds used as perfume and pharmacological agents. A deficiency of squalene (SQ) synthase activity allows yeasts to accumulate an acyclic sesquiterpene alcohol, (E,E)-farnesol, in their cells. Since sterols are essential for the growth of yeasts, a deficiency of SQ synthase activity makes the addition of supplemental sterols to the culture media necessary. To develop a GGOH production method not requiring any supplemental sterols, we overexpressed HMG1 encoding hydroxymethylglutaryl-CoA reductase and the genes of two prenyl diphosphate synthases, ERG20 and BTS1, in Saccharomyces cerevisiae. A prototrophic diploid coexpressing HMG1 and the ERG20-BTS1 fusion accumulated GGOH with neither disruption of the SQ synthase gene nor the addition of any supplemental sterols. The GGOH content on the diploid cultivation in a 5-l jar fermenter reached 138.8 mg/l under optimal conditions.     

Cytosolic phospholipase A2: biochemical properties and physiological roles

Phosphatidylcholine (PC) is a major constituent of biological membranes and a component of serum lipoproteins and pulmonary surfactants. The PC and other glycerophospholipid compositions of membranes change dynamically through stimulus-dependent and independent pathways, principally by the action of two different types of enzymes; phospholipase A2 [EC 3.1.1.4] and acyl-CoA:lysophospholipid acyltransferase [EC 2.3.1.23]. Phospholipase A2 is a key enzyme that catalyzes deacylation of the sn-2 position of glycerophospholipids. This enzyme is critical in the remodeling of membrane lipids and formation of two subclasses of lipid mediators, fatty acid derivatives and lysophospholipids. Among many different subtypes of phospholipase A2 enzymes, we found that cytosolic phospholipase A2alpha (cPLA2alpha) is important in various pathological and physiological responses. Here, we summarize the phenotypes resulting from genetic ablation of cPLA2alpha, and the properties of newly discovered enzymes in the cPLA2 family. Comprehensive analysis of lipid mediators using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is useful for understanding the roles of individual mediators in physiological and pathological processes.     

Effect of some heavy metal ions on copper-induced metallothionein synthesis in the yeast Saccharomyces cerevisiae

Copper-induced metallothionein (MT) synthesis in Saccharomyces cerevisiae was investigated in order to associate this exclusively with Cu²⁺ in vivo, when cultured in nutrient medium containing other heavy metal ions. Expression of the CUP1 promoter/lacZ fusion gene was inhibited by all heavy metal ions tested, especially Cd²⁺ and Mn²⁺. By adding Cd²⁺ and Mn²⁺ at 10 M concentration, the -galactosidase activity decreased by about 80% and 50% of the maximum induction observed with 1 mM CuSO₄, respectively. Furthermore, cell growth was markedly inhibited by combinations of 1 mM-Cu²⁺ and 1 M-Cd²⁺. Therefore, the yeast S. cerevisiae could not rely on MT synthesis as one of the copper-resistance mechanisms, when grown in a Cd²⁺ environment. In contrast, the presence of Mn²⁺ in the nutrient medium showed alleviation rather than growth inhibition by high concentrations of Cu²⁺. The recovery from growth inhibition by Mn²⁺ was due to decreased Cu²⁺ accumulation. Inhibitory concentrations of Co²⁺, Ni²⁺ and Zn²⁺ on expression of the CUP1p/lacZ fusion gene were at least one order of magnitude higher than that of Cd²⁺ and Mn²⁺. These results are discussed in relation to Cu²⁺ transport and Cu-induced MT synthesis in the copper-resistance mechanism of the yeast S. cerevisiae.     

Dual effects on HDL metabolism by cholesteryl ester transfer protein inhibition in HepG2 cells

Cholesteryl ester transfer protein (CETP) promotes reverse cholesterol transport via exchange of cholesteryl ester and triglyceride among lipoproteins. Here, we focused on HDL metabolism during inhibition of CETP expression by using CETP antisense oligodeoxynucleotides (ODNs) in HepG2 cells. CETP secretion was decreased by 70% in mRNA levels and by 52% in mass 20 h after ODNs against CETP were delivered to HepG2 cells. Furthermore, as a consequence of the downregulation of CETP, the expression of scavenger receptor class B type I (SR-BI), an HDL receptor, was also reduced by approximately 50% in mRNA and protein levels, whereas the apolipoprotein A-I (apoA-I) expression and secretion were increased by 30 and 92%, respectively. In a functional study, the selective uptake of (125)I-[(14)C]cholesteryl oleate-labeled HDL(3) was decreased. Cholesterol efflux to apoA-I and HDL(3) was significantly increased by 88 and 37%, respectively. Moreover, the CE levels in cells after antisense treatment were elevated by 20%, which was related to the about twofold increase of cholesterol esterification and increased acyl-CoA:cholesterol acyltransferase 1 mRNA levels. Taken together, these findings suggest that although acute suppression of CETP expression leads to an elevation in cellular cholesterol stores, apoA-I secretion, and cellular cholesterol efflux to apoA-I, the return of HDL-CE to hepatocytes via an SR-BI pathway was inhibited in vitro. Thus antisense inhibition of hepatic CETP expression manifests dual effects: namely, increased formation of HDL and suppression of catabolism of HDL-CE, probably via the SR-BI pathway.     

Molecular interaction and synergistic activation of a promoter by Six,Eya, and Dach proteins mediated through CREB binding protein

Drosophila sine oculis, eyes absent, and dachshund are essential for compound eye formation and form a gene network with direct protein interaction and genetic regulation. The vertebrate homologues of these genes, Six, Eya, and Dach, also form a similar genetic network during muscle formation. To elucidate the molecular mechanism underlying the network among Six, Eya, and Dach, we examined the molecular interactions among the encoded proteins. Eya interacted directly with Six but never with Dach. Dach transactivated a multimerized GAL4 reporter gene by coproduction of GAL4-Eya fusion proteins. Transactivation by Eya and Dach was repressed by overexpression of VP16 or E1A but not by E1A mutation, which is defective for CREB binding protein (CBP) binding. Recruitment of CBP to the immobilized chromatin DNA template was dependent on FLAG-Dach and GAL4-Eya3. These results indicate that CBP is a mediator of the interaction between Eya and Dach. Contrary to our expectations, Dach binds to chromatin DNA by itself, not being tethered by GAL4-Eya3. Dach also binds to naked DNA with lower affinity. The conserved DD1 domain is responsible for binding to DNA. Transactivation was also observed by coproduction of GAL4-Six, Eya, and Dach, indicating that Eya and Dach synergy is relevant when Eya is tethered to DNA through Six protein. Our results demonstrated that synergy is mediated through direct interaction of Six-Eya and through the interaction of Eya-Dach with CBP and explain the molecular basis for the genetic interactions among Six, Eya, and Dach. This work provides fundamental information on the role and the mechanism of action of this gene cassette in tissue differentiation and organogenesis.     

FlgD is a scaffolding protein needed for flagellar hook assembly in Salmonella typhimurium.

FlgD is known to be absolutely required for hook assembly, yet it has not been detected in the mature flagellum. We have overproduced and purified FlgD and raised an antibody against it. By using this antibody, we have detected FlgD in substantial amounts in isolated basal bodies from flgA, flgE, flgH, flgI, flgK, and fliK mutants, in much smaller amounts in those from the wild type and flgL, fliA, fliC, fliD, and fliE mutants, and not at all in those from flgB, flgD, flgG, and flgJ mutants. In terms of the morphological assembly pathway, these results indicate that FlgD is first added to the structure when the rod is completed and is discarded when the hook, having reached its mature length, has the first of the hook-filament junction proteins, FlgK, added to its tip. Immunoelectron microscopy established that FlgD initially is located at the distal end of the rod and eventually is located at the distal end of the hook. Thus, it appears to act as a hook-capping protein to enable assembly of hook protein subunits, much as another flagellar protein, FliD, does for the flagellin subunits of the filament. However, whereas FliD is associated with the filament tip indefinitely, FlgD is only transiently associated with the hook tip; i.e., it acts as a scaffolding protein. When FlgD was added to the culture medium of a flgD mutant, cells gained motility; thus, although the hook cap is normally added endogenously, it can be added exogenously. When culture media were analyzed for the presence of hook protein, it was found only with the flgD mutant and, in smaller amounts, the fliK (polyhook) mutant. Thus, although FlgD is needed for assembly of hook protein, it is not needed for its export.