Development of a quantitative method for determination of the optimal conditions for protoplast isolation from cultured plant cells

An index [kv: average isolation rate of viable protoplast (number/ml min)] was established to evaluate the optimal conditions for protoplast isolation from cultured plant cells. The optimal conditions for protoplasts isolation from Nicotiana tabacum BY2 cultured cells could be determined on the basis of the kv [31.7 × 10³ (number/ml min)]. The colony-forming efficiency of the protoplasts was about 46%. The optimal conditions for protoplasts isolation from Catharanthus roseus [kv = 38.1 × 10³ (number/ml min)] and Wasabia japonica [kv = 14.2 × 10³ (number/ml min)] cultured cells could also be determined. Furthermore, a method for rapid regenerating cell wall of protoplast in liquid culture using alginate gel containing locust bean gum was developed.     

Molecular and catalytic properties of 2,4'-dihydroxyacetophenone dioxygenase from Burkholderia sp. AZ11

The gene dad encoding 2,4'-dihydroxyacetophenone (DHAP) dioxygenase was cloned from Burkholderia sp. AZ11. The initiation codon GTG was converted to ATG for high-level expression of the enzyme in Escherichia coli. The enzyme was moderately thermostable, and the recombinant enzyme was briefly purified. The enzyme (M(r)=90 kDa) was a homotetramer with a subunit M(r) of 23 kDa. It contained 1.69 mol of non-heme iron, and had a dark gray color. On anaerobic incubation of it with DHAP, the absorption at around 400 nm increased due to the formation of an enzyme-DHAP complex. Multiple sequence alignment suggested that His77, His79, His115, and Glu96 in the cupin fold were possible metal ligands. The apparent K(m) for DHAP and the apparent V(max) were estimated to be 1.60 μM and 6.28 μmol/min/mg respectively. 2-Hydroxyacetophenone was a poor substrate. CuCl(2) and HgCl(2) strongly inhibited the enzyme, while FeSO(4) weakly activated it.     

Systemic PPARγ Deletion Impairs Circadian Rhythms of Behavior and Metabolism

Compelling evidence from both human and animal studies suggests a physiological link between the circadian rhythm and metabolism but the underlying mechanism is still incompletely understood. We examined the role of PPARγ, a key regulator of energy metabolism, in the control of physiological and behavioral rhythms by analyzing two strains of whole-body PPARγ null mouse models. Systemic inactivation of PPARγ was generated constitutively by using Mox2-Cre mice (MoxCre/flox) or inducibly by using the tamoxifen system (EsrCre/flox/TM). Circadian variations in oxygen consumption, CO(2) production, food and water intake, locomotor activity, and cardiovascular parameters were all remarkably suppressed in MoxCre/flox mice. A similar phenotype was observed in EsrCre/flox/TM mice, accompanied by impaired rhythmicity of the canonical clock genes in adipose tissues and liver but not skeletal muscles or the kidney. PPARγ inactivation in isolated preadipocytes following exposure to tamoxifen led to a similar blockade of the rhythmicity of the clock gene expression. Together, these results support an essential role of PPARγ in the coordinated control of circadian clocks and metabolic pathways.     

New liquid chromatography method combining thermo-responsive material and inductive heating via alternating magnetic field

In this study, we examined the feasibility of a novel liquid chromatography technique that combines temperature-responsive polymeric materials with inductive heating via an alternating magnetic field (AMF). We considered the following components of the technique: (i) the preparation of composite materials of magnetite and silica, (ii) their heating behavior under the AMF, (iii) the conjugation of temperature-responsive polymers for the packing materials, and (iv) the elution profiles of the model compounds in the AMF. The results showed that we could influence the elution of the model compounds by AMF induction heating generated by the surrounding coil.     

Expression and characterization of the Streptomyces coelicolor serine/threonine protein kinase PkaD

We identified and characterized the gene encoding a new eukaryotic-type protein kinase from Streptomyces coelicolor A3(2) M145. PkaD, consisting of 598 amino acid residues, contained the catalytic domain of eukaryotic protein kinases in the N-terminal region. A hydrophobicity plot indicated the presence of a putative transmembrane spanning sequence downstream of the catalytic domain, suggesting that PkaD is a transmembrane protein kinase. The recombinant PkaD was found to be phosphorylated at the threonine and tyrosine residues. In S. coelicolor A3(2), pkaD was transcribed as a monocistronic mRNA, and it was expressed constitutively throughout the life cycle. Disruption of chromosomal pkaD resulted in a significant loss of actinorhodin production. This result implies the involvement of pkaD in the regulation of secondary metabolism.     

Propionate sensor using coenzyme-A transferase and acyl-CoA oxidase

We developed an amperometric propionate sensor using comprised of two recombinant enzymes, propionate coenzyme A CoA transferase from Clostridium propionicum and short-chain acyl-CoA oxidase from Arabidopsis thaliana. Response current increased linearly with increase in propionate concentration from 10 microM to 100 microM. The detection limit was 10 microM propionate.     

Comparison of metabolite production capability indices generated by network analysis methods

A framework of constraint-based reconstruction and analysis (COBRA) is used for modeling large-scale metabolic networks. In COBRA, extreme pathway and optimization analyses are commonly used to study the properties of networks. While the results of both methods are completely consistent, extreme pathway analysis is considered to be better because of its wider representational ability. In this study, we assessed these two methods by computational knockout experiments. We examined a simple pathway model and found that the extreme pathway method led to misguided conclusions in specific cases, while optimization analysis calculated the correct knockout effects. We also investigated the Escherichia coli metabolic pathway model, and found that these methods result in inconsistent interpretations of the network properties. IN CONCLUSION: it has been claimed that these two methods result in the same producible metabolites, but we found a difference in individual results for a biological pathway. Our results could provide helpful guidance for when to use the methods, particularly extreme pathway analysis.     

Effect of heat-generated product from uronic acids on the physiological activities of microbial cells and its application

Filtered samples of monogalacturonic (GA) and monoglucuronic acids (GL) that were prepared using millipore filter (pore size=0.2 microm) slightly inhibited the growth of Escherichia coli while the autoclaved (at 121 degrees C for 20 min) samples of GA and GL completely inhibited the growth of E. coli. The most effective substance generated upon autoclave treatment was isolated and characterized as trans-4,5-dihydroxy-2-cyclopenten-1-one (DHCP). The optimal conditions for DHCP generation were also established by autoclaving GA (pH 2.3) at 121 degrees C for 3h. DHCP completely inhibited the growth of E. coli. However, the growth of E. coli was restored when superoxide dismutase and catalase were added to the culture broth that contained DHCP. It was thought that DHCP might have induced the release of active oxygen, which resulted in the inhibition of microbial growth. In the case of gram-positive bacteria (Bacillus cereus, Bacillus subtilis and Staphylococcus aureus) and yeast (Saccharomyces cerevisiae and Candida brassicae), DHCP inhibited the cell growth. Based on our results, methods for preparation of food preservatives that contained pectin degraded products (oligo-galacturonic acid and monogalacturonic acid) and DHCP were developed. The preservatives were very effective in inhibiting the growth of E. coli and S. cerevisiae.     

Interaction of mastoparan with membranes studied by 1H-NMR spectroscopy in detergent micelles and by solid-state 2H-NMR and 15N-NMR spectroscopy in oriented lipid bilayers.

Several complementary NMR approaches were used to study the interaction of mastoparan, a 14-residue peptide toxin from wasp venom, with lipid membranes. First, the 3D structure of mastoparan was determined using 1H-NMR spectroscopy in perdeuterated (SDS-d25) micelles. NOESY experiments and distance geometry calculations yielded a straight amphiphilic alpha-helix with high-order parameters, and the chemical shifts of the amide protons showed a characteristic periodicity of 3-4 residues. Secondly, solid-state 2H-NMR spectoscopy was used to describe the binding of mastoparan to lipid bilayers, composed of headgroup-deuterated dimyristoylglycerophosphocholine (DMPC-d4) and dimyristoylphosphatidylglycerol (DMPG). By correlating the deuterium quadrupole splittings of the alpha-segments and beta-segments, it was possible to differentiate the electrostatically induced structural response of the choline headgroup from dynamic effects induced by the peptide. A partial phase separation was observed, leading to a DMPG-rich phase and a DMPG-depleted phase, each containing some mastoparan. Finally, the insertion and orientation of a specifically 15N-labeled mastoparan (at position Ala10) in the bilayer environment was investigated by solid-state 15N-NMR spectroscopy, using macroscopically oriented samples. Two distinct orientational states were observed for the mastoparan helix, namely an in-plane and a trans-membrane alignment. The two populations of 90% in-plane and 10% trans-membrane helices are characterized by a mosaic spread of +/- 30 degrees and +/- 10 degrees, respectively. The biological activity of mastoparan is discussed in terms of a pore-forming model, as the peptide is known to be able to induce nonlamellar phases and facilitate a flip-flop between the monolayers.     

Distribution of alginate oligomers in Catharanthus roseus and Wasabia japonica cells cultured in a medium containing alginate oligomers

Distribution of alginate oligomers (AO) which are endogenous elicitor-like substances, in cultured plant cells were investigated by using AO conjugated with monopotassium 7-amino-1,3-naphthalenedisulfonate (ANDS). When AO-ANDS was added at 0.5 g l^–1 to the Catharanthus roseus cell culture, it adhered to the cells as observed by fluorescence microscopy. Using protoplasts of C. roseus, AO-ANDS was found not only in the cell walls but also in the cell membrane and cytoplasm. When C. roseus was cultivated in a medium containing oligo-galacturonic acids, as an endogenous elicitor, this was also found in the cell wall, cell membrane and cytoplasm of C. roseus cells. Similar results were also obtained with Wasabia japonica cells.