Metabolome analysis of photosynthesis and the related primary metabolites in the leaves of transgenic rice plants with increased or decreased Rubisco content

Because the comprehensive effects on metabolism by genetic manipulation of leaf Rubisco content are unknown, metabolome analysis was carried out on transgenic rice plants with increased or decreased Rubisco content using the capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) technique. In RBCS-sense plants, an increase in Rubisco content did not improve light-saturated photosynthesis. Glyceraldehyde 3-phosphate and sedoheputulose 7-phosphate levels increased, but ribulose bisphosphate (RuBP), ATP and ADP levels were not affected. It is considered from these results that RuBP regeneration independent of ATP supply became a bottleneck for photosynthesis. In RBCS-antisense plants, a decline in Rubisco content decreased photosynthesis with a substantial accumulation of RuBP. ATP and ADP levels also increased and were associated with increases in the diphosphate and triphosphate compounds of other nucleosides. These results imply that a decline in Rubisco content slowed down the Calvin cycle and that the resultant excess energy of ATP was transferred to other nucleoside diphosphates and triphosphates. The levels of amino acids tended to decline in RBCS-sense plants and increase in RBCS-antisense plants, probably reflecting the demand for Rubisco synthesis. Starch and carbohydrate levels decreased only in RBCS-antisense plants. Thus, genetic manipulation of Rubisco contents widely affected C and N metabolism in rice.     

A novel convergent method for the synthesis of α-acyl-γ-hydroxylactams and its application in the total synthesis of PI-090 and 091

A novel convergent method for the synthesis of α-acyl-γ-hydroxylactams utilizing the aldol reaction of N-Boc-protected γ-methoxylactams was developed. As the first application of this method for the synthesis of biologically active natural products, the total synthesis of platelet aggregation inhibitors PI-090 and PI-091 were also investigated and successfully achieved.     

Dom34:hbs1 plays a general role in quality-control systems by dissociation of a stalled ribosome at the 3' end of aberrant mRNA

Translation arrest leads to an endonucleolytic cleavage of mRNA that is termed no-go decay (NGD). It has been reported that the Dom34:Hbs1 complex stimulates this endonucleolytic cleavage of mRNA induced by translation arrest in vivo and dissociates subunits of a stalled ribosome in vitro. Here we report that Dom34:Hbs1 dissociates the subunits of a ribosome that is stalled at the 3' end of mRNA in vivo, and has a crucial role in both NGD and nonstop decay. Dom34:Hbs1-mediated dissociation of a ribosome that is stalled at the 3' end of mRNA is required for degradation of a 5'-NGD intermediate. Dom34:Hbs1 facilitates the decay of nonstop mRNAs from the 3' end by exosomes and is required for the complete degradation of nonstop mRNA decay intermediates. We propose that Dom34:Hbs1 stimulates degradation of the 5'-NGD intermediate and of nonstop mRNA by dissociating the ribosome that is stalled at the 3' end of the mRNA.     

Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells

The severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 protein has unique biological functions that have not been described in the viral proteins of any RNA viruses; expressed SARS-CoV nsp1 protein has been found to suppress host gene expression by promoting host mRNA degradation and inhibiting translation. We generated an nsp1 mutant (nsp1-mt) that neither promoted host mRNA degradation nor suppressed host protein synthesis in expressing cells. Both a SARS-CoV mutant virus, encoding the nsp1-mt protein (SARS-CoV-mt), and a wild-type virus (SARS-CoV-WT) replicated efficiently and exhibited similar one-step growth kinetics in susceptible cells. Both viruses accumulated similar amounts of virus-specific mRNAs and nsp1 protein in infected cells, whereas the amounts of endogenous host mRNAs were clearly higher in SARS-CoV-mt-infected cells than in SARS-CoV-WT-infected cells, in both the presence and absence of actinomycin D. Further, SARS-CoV-WT replication strongly inhibited host protein synthesis, whereas host protein synthesis inhibition in SARS-CoV-mt-infected cells was not as efficient as in SARS-CoV-WT-infected cells. These data revealed that nsp1 indeed promoted host mRNA degradation and contributed to host protein translation inhibition in infected cells. Notably, SARS-CoV-mt infection, but not SARS-CoV-WT infection, induced high levels of beta interferon (IFN) mRNA accumulation and high titers of type I IFN production. These data demonstrated that SARS-CoV nsp1 suppressed host innate immune functions, including type I IFN expression, in infected cells and suggested that SARS-CoV nsp1 most probably plays a critical role in SARS-CoV virulence.     

Near-infrared fluorescent labeled peptosome for application to cancer imaging

Nonionic amphiphilic copolypeptides, which were composed of hydrophilic poly(sarcosine) and hydrophobic poly(gamma-methyl L-glutamate) blocks, were synthesized with varying chain lengths of the blocks. The polypeptides having a suitable hydrophilic and hydrophobic balance were found to form vesicular assemblies of 100 nm size in buffer, which was evidenced by the TEM observation, the DLS analysis, and the encapsulation experiment. The genuine peptide vesicles, peptosomes, were labeled with a near-infrared fluorescence (NIRF) probe. In vivo retention in blood experiment showed long circulation of the peptosome in rat blood as stable as the PEGylated liposome. NIRF imaging of a small cancer on mouse by using the peptosome as a nanocarrier was successful due to the EPR effect of the peptosome. Peptosome is shown here as a novel excellent nanocarrier for molecular imaging.     

Effects of Protoceratium reticulatum yessotoxin on feeding rates of Acartia hudsonica: A bioassay using artificial particles coated with purified toxin

Paralytic shellfish toxins produced by dinoflagellates are known to deter copepod grazing. Dinoflagellate species, including Protoceratium reticulatum, also produce disulfated polyether yessotoxins that were previously referred to as diarrheic shellfish toxins. However, the role of yessotoxins in predator–prey relationships is not yet clear. In the present study, the effects of purified yessotoxin (YTX) on feeding activities of Acartia hudsonica (Copepoda, Calanoida) were experimentally investigated. Polystyrene fluorescent microspheres (10 μm in diameter) colored bright blue or yellow-green were coated with cell extracts of P. reticulatum that do not produce yessotoxins. The bright blue microspheres were further coated with YTX, and the yellow-green microspheres were used as the reference. The microspheres were then given to the copepods separately or in combination to measure clearance rates and feeding selectivity. A. hudsonica was found to feed on the yellow-green microspheres without YTX at twice the rate of the bright blue microspheres with YTX. We also confirmed that microsphere color per se did not affect the feeding rates. The bright blue microspheres adsorbed 1.8–43.3 pg of YTX per microsphere, which is similar to the cell-specific yessotoxin content of toxic P. reticulatum found in natural environments. These results suggest that production of yessotoxin is advantageous for P. reticulatum by deterring predation by copepods.     

Metabolic fate of L-lactaldehyde derived from an alternative L-rhamnose pathway

Fungal Pichia stipitis and bacterial Azotobacter vinelandii possess an alternative pathway of L-rhamnose metabolism, which is different from the known bacterial pathway. In a previous study (Watanabe S, Saimura M & Makino K (2008) Eukaryotic and bacterial gene clusters related to an alternative pathway of non-phosphorylated L-rhamnose metabolism. J Biol Chem283, 20372-20382), we identified and characterized the gene clusters encoding the four metabolic enzymes [L-rhamnose 1-dehydrogenase (LRA1), L-rhamnono-gamma-lactonase (LRA2), L-rhamnonate dehydratase (LRA3) and l-2-keto-3-deoxyrhamnonate aldolase (LRA4)]. In the known and alternative L-rhamnose pathways, L-lactaldehyde is commonly produced from l-2-keto-3-deoxyrhamnonate and L-rhamnulose 1-phosphate by each specific aldolase, respectively. To estimate the metabolic fate of L-lactaldehyde in fungi, we purified L-lactaldehyde dehydrogenase (LADH) from P. stipitis cells L-rhamnose-grown to homogeneity, and identified the gene encoding this enzyme (PsLADH) by matrix-assisted laser desorption ionization-quadruple ion trap-time of flight mass spectrometry. In contrast, LADH of A. vinelandii (AvLADH) was clustered with the LRA1-4 gene on the genome. Physiological characterization using recombinant enzymes revealed that, of the tested aldehyde substrates, L-lactaldehyde is the best substrate for both PsLADH and AvLADH, and that PsLADH shows broad substrate specificity and relaxed coenzyme specificity compared with AvLADH. In the phylogenetic tree of the aldehyde dehydrogenase superfamily, PsLADH is poorly related to the known bacterial LADHs, including that of Escherichia coli (EcLADH). However, despite its involvement in different L-rhamnose metabolism, AvLADH belongs to the same subfamily as EcLADH. This suggests that the substrate specificities for L-lactaldehyde between fungal and bacterial LADHs have been acquired independently.     

Involvement of Sema4A in the progression of experimental autoimmune myocarditis

Dilated cardiomyopathy often results from autoimmunity triggered by microbial infections during myocarditis. However, it remains unclear how immunological disorders are implicated in pathogenesis of autoimmune myocarditis. Here, we demonstrated that Sema4A, a class IV semaphorin, plays key roles in experimental autoimmune myocarditis (EAM). Dendritic cells pulsed with myosin heavy chain-α peptides induced severe myocarditis in wild-type mice, but not in Sema4A-deficient mice. In adoptive transfer experiments, CD4⁺ T-cells from wild-type mice induced severe myocarditis, while CD4⁺ T-cells from Sema4A-deficient mice exhibited considerably attenuated myocarditis. Our results indicated that Sema4A is critically involved in EAM by regulating differentiation of T-cells.     

Changes in the synthesis of rubisco in rice leaves in relation to senescence and N influx

BACKGROUND AND AIMS: The amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) synthesized in a leaf is closely correlated with N influx into the leaf throughout its lifetime. Rubisco synthesis and N influx are most active in the young leaf during expansion, but are very limited in the senescent leaf. However, it is not established whether Rubisco synthesis can be observed if N influx is increased, even in a very senescent leaf. This study first investigated changes in the relationships between rbcS and rbcL mRNA contents and Rubisco synthesis per unit of leaf mass with leaf senescence. Next, leaves were removed during late senescence, to examine whether Rubisco synthesis is re-stimulated in very senescent leaves by an increase in N influx. METHODS: Different N concentrations (1 and 4 mm) were supplied to Oryza sativa plants at the early (full expansion), middle and late stages (respectively 8 and 16 d after full expansion) of senescence of the eighth leaf. To enhance N influx into the eighth leaf 16 d after full expansion, all leaf blades on the main stem, except for the eighth leaf, and all tillers were removed and plants received 4 mm N (removal treatment). KEY RESULTS: Rubisco synthesis, rbcS and rbcL mRNAs and the translational efficiencies of rbcS and rbcL mRNAs decreased with leaf senescence irrespective of N treatments. However, in the removal treatment at the late stage, they increased more strongly with an increase in N influx than in intact plants. CONCLUSIONS: Although Rubisco synthesis and rbcS and rbcL mRNAs decrease with leaf senescence, leaves at the late stage of senescence have the potential actively to synthesize Rubisco with an increase in N influx.