Overexpression of BUNDLE SHEATH DEFECTIVE 2 improves the efficiency of photosynthesis and growth in Arabidopsis

Bundle Sheath Defective 2, BSD2, is a stroma‐targeted protein initially identified as a factor required for the biogenesis of ribulose 1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) in maize. Plants and algae universally have a homologous gene for BSD2 and its deficiency causes a RuBisCO‐less phenotype. As RuBisCO can be the rate‐limiting step in CO₂ assimilation, the overexpression of BSD2 might improve photosynthesis and productivity through the accumulation of RuBisCO. To examine this hypothesis, we produced BSD2 overexpression lines in Arabidopsis. Compared with wild type, the BSD2 overexpression lines BSD2ox‐2 and BSD2ox‐3 expressed 4.8‐fold and 8.8‐fold higher BSD2 mRNA, respectively, whereas the empty‐vector (EV) harbouring plants had a comparable expression level. The overexpression lines showed a significantly higher CO₂ assimilation rate per available CO₂ and productivity than EV plants. The maximum carboxylation rate per total catalytic site was accelerated in the overexpression lines, while the number of total catalytic sites and RuBisCO content were unaffected. We then isolated recombinant BSD2 (rBSD2) from E. coli and found that rBSD2 reduces disulfide bonds using reductants present in vivo, for example glutathione, and that rBSD2 has the ability to reactivate RuBisCO that has been inactivated by oxidants. Furthermore, 15% of RuBisCO freshly isolated from leaves of EV was oxidatively inactivated, as compared with 0% in BSD2‐overexpression lines, suggesting that the overexpression of BSD2 maintains RuBisCO to be in the reduced active form in vivo. Our results demonstrated that the overexpression of BSD2 improves photosynthetic efficiency in Arabidopsis and we conclude that it is involved in mediating RuBisCO activation.     

Nocturnal uptake and assimilation of nitrogen dioxide by C3 and CAM plants

In order to investigate nocturnal uptake and assimilation of NO2 by C3 and crassulacean acid metabolism (CAM) plants, they were fumigated with 4 microl l(-1) 15N-labeled nitrogen dioxide (NO2) for 8 h. The amount of NO2 and assimilation of NO2 by plants were determined by mass spectrometry and Kjeldahl-nitrogen based mass spectrometry, respectively. C3 plants such as kenaf (Hibiscus cannabinus), tobacco (Nicotiana tabacum) and ground cherry (Physalis alkekengi) showed a high uptake and assimilation during daytime as high as 1100 to 2700 ng N mg(-1) dry weight. While tobacco and ground cherry strongly reduced uptake and assimilation of NO2 during nighttime, kenaf kept high nocturnal uptake and assimilation of NO2 as high as about 1500 ng N mg(-1) dry weight. Stomatal conductance measurements indicated that there were no significant differences to account for the differences in the uptake of NO2 by tobacco and kenaf during nighttime. CAM plants such as Sedum sp., Kalanchoe blossfeldiana (kalanchoe) and Aloe arborescens exhibited nocturnal uptake and assimilation of NO2. However, the values of uptake and assimilation of NO2 both during daytime and nighttime was very low (at most about 500 ng N mg(-1) dry weight) as compared with those of above mentioned C3 plants. The present findings indicate that kenaf is an efficient phytoremediator of NO2 both during daytime and nighttime.     

Novel metabolism of nitrogen in plants

Our previous study showed that approximately one-third of the nitrogen of 15N-labeled NO2 taken up into plants was converted to a previously unknown organic nitrogen (hereafter designated UN) that was not recoverable by the Kjeldahl method (Morikawa et al., 2004). In this communication, we discuss metabolic and physiological relevance of the UN based on our newest experimental results. All of the 12 plant species were found to form UN derived from NO2 (about 10-30% of the total nitrogen derived from NO2). The UN was formed also from nitrate nitrogen in various plant species. Thus, UN is a common metabolite in plants. The amount of UN derived from NO2 was greatly increased in the transgenic tobacco clone 271 (Vaucheret et al., 1992) where the activity of nitrite reductase is suppressed less than 5% of that of the wild-type plant. On the other hand, the amount of this UN was significantly decreased by the overexpression of S-nitrosoglutathione reductase (GSNOR). These findings strongly suggest that nitrite and other reactive nitrogen species are involved in the formation of the UN, and that the UN-bearing compounds are metabolizable. A metabolic scheme for the formation of UN-bearing compounds was proposed, in which nitric oxide and peroxynitrite derived from NO2 or endogenous nitrogen oxides are involved for nitrosation and/or nitration of organic compounds in the cells to form nitroso and nitro compounds, including N-nitroso and S-nitroso ones. Participation of non-symbiotic haemoglobin bearing peroxidase-like activity (Sakamoto et al., 2004) and GSNOR (Sakamoto et al., 2002) in the metabolism of the UN was discussed. The UN-bearing compounds identified to date in the extracts of the leaves of Arabidopsis thaliana fumigated with NO2 include a delta2-1,2,3-thiadiazoline derivative (Miyawaki et al., 2004) and 4-nitro-beta-carotene.     

Purification and characterization of a soluble recombinant human ST6Gal I functionally expressed in Escherichia coli

A soluble and active form of recombinant human ST6Gal I was expressed in Escherichia coli. The gene encoding the soluble form of ST6Gal I lacking the membrane and cytosolic regions was introduced into a bacterial expression vector, pMAL-p2X, fused in frame with a maltose-binding protein (MBP) tag. Low-temperature cultivation at 13C during IPTG-induction significantly improved both solubility and MBP-tagging of the recombinant enzyme expressed in bacteria. The supernatant prepared by disruption of the cells demonstrated sialic acid transfer activity to both an oligosaccharide and a glycoprotein, asialofetuin, indicating that the enzyme expressed in bacteria is soluble and active. The MBP-tagged enzyme was efficiently purified by a combination of cation-exchange column and amylase-conjugated agarose column chromatography. The purified recombinant enzyme exerted enzymatic activity even in the absence of detergents in the reaction mixture. Acceptor substrate specificity of the enzyme was marginally different from that of rat liver ST6Gal I. These observations suggest that membrane and cytosolic regions of ST6Gal I may affect the properties of the enzyme. The purified recombinant enzyme was applied to convert desialylated fetuin to resialylated fetuin. Lectin blotting demonstrated that resialylated fetuin possesses a single Neu5Ac 2-6 residue. The resialylated fetuin efficiently blocked hemagglutination induced by influenza virus strain A/Memphis/1/71 (H3N2), indicating that resialylated carbohydrate chains on the protein are so active as to competitively inhibit virus-receptor interaction. In conclusion, soluble recombinant ST6Gal I obtained using our bacterial expression system is a valuable tool to investigate the molecular mechanisms of biological and pathological interactions mediated via carbohydrates. Published in 2005.The authors contributed equally to this work.     

Practical resolution system for DL-pantoyl lactone using the lactonase from Fusarium oxysporum

We developed an enzymatic resolution system for DL-pantoyl lactone that uses immobilized mycelia of Fusarium oxysporum, which produce a lactone-hydrolyzing enzyme (lactonase). The lactonase catalyzes the stereospecific hydrolysis of D-pantoyl lactone. One hundred eighty repeated batch reactions (total reaction time, 3780 h) were made with mycelia entrapped in calcium alginate gels as the catalyst, in the presence of 90 mM CaCl2. With a 300 gl(-1)DL-pantoyl lactone solution as the substrate, the hydrolysis rate for DL-pantoyl lactone was > 40% and the optical purity of D-pantoic acid was 90% enantiomer excess. Immobilized mycelia retained 70% of their initial lactonase activity, even after 180 batch reactions. The estimated half-life of the lactonase activity of the immobilized mycelia was 6000 h, which is 35 times higher than that of the free mycelia. The process has been exploited commercially since 1999.     

Crystallization of halorhodopsin from Halobacterium sp. shark

The chloride-ion-pumping channel, halorhodopsin from Halobacterium sp. shark was detergent-solubilized and 3-D crystallized. Proteins were solubilized using the nonionic detergent n-octyl-beta-D-glucoside and were crystallized as thin-plate crystals with polyethylene glycol 4000 as a precipitant. The crystals belong to the space group P4(1)2(1)2 with unit-cell dimensions a=b=74.5 A and c=138.6 A. The diffraction pattern was slightly anisotropic. The best ordered crystal diffracted up to 3.3 A resolution along c axis with synchrotron radiation.     

Step-size is determined by neck length in myosin V

The highly processive motor, myosin V, has an extremely long neck containing six calmodulin-binding IQ motifs that allows it to take multiple 36 nm steps corresponding to the pseudo-repeat of actin. To further investigate how myosin V moves processively on actin filaments, we altered the length of the neck by adding or deleting IQ motifs in myosin constructs lacking the globular tail domain. These myosin V IQ mutants were fluorescently labeled by exchange of a single Cy3-labeled calmodulin into the neck region of one head. We measured the step-size of these individual IQ mutants with nanometer precision and subsecond resolution using FIONA. The step-size was proportional to neck length for constructs containing 2, 4, 6, and 8 IQ motifs, providing strong support for the swinging lever-arm model of myosin motility. In addition, the kinetics of stepping provided additional support for the hand-over-hand model whereby the two heads alternately assume the leading position. Interestingly, the 8IQ myosin V mutant gave a broad distribution of step-sizes with multiple peaks, suggesting that this mutant has many choices of binding sites on an actin filament. These data demonstrate that the step-size of myosin V is affected by the length of its neck and is not solely determined by the pseudo-repeat of the actin filament.     

Statins downregulate ATP-binding-cassette transporter A1 gene expression in macrophages

The ATP-binding-cassette transporter A1 (ABCA1) plays an essential role in cellular cholesterol efflux and helps prevent macrophages from becoming foam cells. The statins are widely used as cholesterol-lowering agents and have other anti-atherogenic actions. We tested the effects of four different statins (fluvastatin, atorvastatin, simvastatin, and lovastatin) on ABCA1 expression in macrophages in vitro. The statins suppressed ABCA1 mRNA expression in RAW246.7 and THP-1 macrophage cell lines and in mouse peritoneal macrophages. The effect was time- and dose-dependent and was abolished by the addition of the post-reductase product, mevalonate. These findings imply that there is a possible modulation of the well-known beneficial effects of the statins on the reverse cholesterol transport pathway.     

Both the sequence and length of the C terminus of PEN-2 are critical for intermolecular interactions and function of presenilin complexes

Presenilin 1 or presenilin 2, nicastrin, APH-1, and PEN-2 form high molecular weight complexes that play a pivotal role in the cleavage of various Type I transmembrane proteins, including the beta-amyloid precursor protein. The specific function of PEN-2 is unclear. To explore its function and intermolecular interactions, we conducted deletion and mutagenesis studies on a series of conserved residues at the C terminus of PEN-2. These studies suggest that: 1) both the presence and amino acid sequence of the conserved DYLSF domain at the C terminus of PEN-2 (residues 90-94) is critical for binding PEN-2 to other components in the presenilin complex and 2) the overall length of the exposed C terminus is critical for functional gamma-secretase activity.