Transgenic tobacco plants producing caffeine: a potential new strategy for insect pest control

Caffeine (1,3,7–trimethylxanthine) is one of the most widely used plant secondary metabolites, primarily as a stimulant and an ingredient in drugs. In nature, caffeine is believed to function in chemical defense, acting as an antiherbivory and allelopathic agent, and therefore it might be employed to protect agriculturally important crop plants. In coffee plants, caffeine is synthesized from the precursor xanthosine in four steps, three N-methylations and removal of ribose. We had previously isolated genes encoding three distinct N-methyltransferases, and we demonstrated production of recombinant enzymes that yielded caffeine in in vitro reconstitution experiments. When these caffeine biosynthetic pathway genes were simultaneously expressed in tobacco plants (Nicotiana tabacum), caffeine was successfully produced up to 5 μg/g fresh weight in leaves. The leaves were unpalatable to tobacco cutworms (Spodoptera litura). This repellent action appeared to be more widely␣applicable to lepidopteran caterpillars as observed with small white (Pieris rapae) fed on Chinese cabbages that had been top-treated with caffeine. Our recent results suggest a novel approach to strengthen anti-herbivore traits by producing caffeine in crop plants.     

Curcumin dramatically enhances retinoic acid-induced superoxide generating activity via accumulation of p47-phox and p67-phox proteins in U937 cells

The membrane bound cytochrome b558 composed of large gp91-phox and small p22-phox subunits, and cytosolic proteins p40-, p47- and p67-phox are important components of superoxide ()-generating system in phagocytes and B lymphocytes. A lack of this system in phagocytes is known to cause serious life-threatening infections. Here, we describe that curcumin, a polyphenol responsible for the yellow color of curry spice turmeric, dramatically activates the -generating system during retinoic acid (RA)-induced differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. When U937 cells were cultured in the presence of RA and curcumin, the -generating activity increased more than 4-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and curcumin slightly enhanced gene expressions of the five components compared with those of the RA-treatment only. On the other hand, immunoblot analysis revealed that co-treatment with RA and curcumin caused remarkable accumulation of protein levels of p47-phox (to 7-fold) and p67-phox (to 4-fold) compared with those of the RA-treatment alone. These results suggested that curcumin dramatically enhances RA-induced -generating activity via accumulation of cytosolic p47-phox and p67-phox proteins in U937 cells. Therefore, it should have the potential as an effective modifier in therapy of leukemia and/or as an immunopotentiator.     

Substitution of Glu122 by Glutamine Revealed the Function of the Second Water Molecule as a Proton Donor in the Binuclear Metal Enzyme Creatininase

Creatininase is a binuclear zinc enzyme and catalyzes the reversible conversion of creatinine to creatine. It exhibits an open-closed conformational change upon substrate binding, and the differences in the conformations of Tyr121, Trp154, and the loop region containing Trp174 were evident in the enzyme-creatine complex when compared to those in the ligand-free enzyme. We have determined the crystal structure of the enzyme complexed with a 1-methylguanidine. All subunits in the complex existed as the closed form, and the binding mode of creatinine was estimated. Site-directed mutagenesis revealed that the hydrophobic residues that show conformational change upon substrate binding are important for the enzyme activity.We propose a catalytic mechanism of creatininase in which two water molecules have significant roles. The first molecule is a hydroxide ion (Wat1) that is bound as a bridge between the two metal ions and attacks the carbonyl carbon of the substrate. The second molecule is a water molecule (Wat2) that is bound to the carboxyl group of Glu122 and functions as a proton donor in catalysis. The activity of the E122Q mutant was very low and it was only partially restored by the addition of ZnCl₂ or MnCl₂. In the E122Q mutant, k_cat is drastically decreased, indicating that Glu122 is important for catalysis. X-ray crystallographic study and the atomic absorption spectrometry analysis of the E122Q mutant-substrate complex revealed that the drastic decrease of the activity of the E122Q was caused by not only the loss of one Zn ion at the Metal1 site but also a critical function of Glu122, which most likely exists for a proton transfer step through Wat2.     

Fish habitat rehabilitation using wood in the world

To provide river managers and researchers with practical knowledge about fish rehabilitation, various studies of fish habitat rehabilitation that used wood were reviewed. The review focuses on fish responses, wood installation methods, and geomorphic features of the rehabilitation sites. Most studies were conducted in moderately sized (small and medium) streams with relatively high bed gradients and aimed to improve the habitats of salmonid species. In this stream type, structures spanning the full (log dam) and partial (log deflector) width of the river were most common, and wood structures that created pools and covers were successful in improving fish habitat. Some projects were conducted in moderately sized low-gradient streams, in which wooden devices used to create instream cover were effective for fish assemblages. There were few studies in other aquatic ecosystems. However, well-designed large wood structures, known as engineered log jams, were used in rehabilitation projects for large rivers. In slack-water or lentic systems such as side-channels, estuaries, and reservoirs, small and large wood structures that created cover were used to improve habitat for many fish species. For successful fish habitat rehabilitation projects, the hydrogeomorphic conditions of rehabilitation sites should be carefully examined to avoid physical failure of wood structures. Although artificial wood structures can be used to improve fish habitat in various aquatic ecosystems, they should be considered to be a complementary or interim habitat enhancement technique. The recovery of natural dynamic processes at the watershed scale is the ultimate target of restoration programs.     

Involvement of reactive nitrogen oxides for acquisition of metastatic properties of benign tumors in a model of inflammation-based tumor progression.

The cells of a weakly tumorigenic and non-metastatic murine fibrosarcoma (QR-32) are converted into highly malignant tumors (acquiring metastatic potential) once they have grown in vivo after being co-implanted with gelatin sponge which induces inflammation. In the present study, we examined whether nitric oxide (NO) is involved in the inflammation-based tumor progression by administrating a specific inhibitor to inducible nitric oxide synthase, aminoguanidine (AG). First, we co-implanted 1 x 10(5) QR-32 cells with gelatin sponge (10 x 5 x 3 mm piece) into a subcutaneous space in C57BL6 mice. Administration of AG in drinking water (1%) had started 2 days before the tumor implantation and continued until the termination of the experiment. The incidence of tumor formation and the tumor growth did not differ between AG-treated group and -untreated group. On day 28, we excised the arising tumors to establish culture cell lines for evaluation of their acquisition of metastatic phenotype in other normal mice. Metastasis incidence and the number of metastatic colonies were significantly reduced in the tumor cell lines obtained from AG-treated mice compared to those from non-treated mice (p < 0.05). Immunohistochemical analysis demonstrated that inducible nitric oxide synthase and nitrotyrosine in the inflamed lesion were reduced in the AG-administered mice. However, intensity of 8-hydroxy-2-deoxyguanosine was not different between the groups. These results showed that nitric oxide and its reactive nitrogen oxide species cooperatively play a pivotal role in the progression of benign tumor cells in inflamed lesions.