A tRNA aminoacylation system for non-natural amino acids based on a programmable ribozyme

The ability to recognize tRNA identities is essential to the function of the genetic coding system. In translation aminoacyl-tRNA synthetases (ARSs) recognize the identities of tRNAs and charge them with their cognate amino acids. We show that an in vitro evolved ribozyme can also discriminate between specific tRNAs, and can transfer amino acids to the 3' ends of cognate tRNAs. The ribozyme interacts with both the CCA-3' terminus and the anticodon loop of tRNA(fMet), and its tRNA specificity is controlled by these interactions. This feature allows us to program the selectivity of the ribozyme toward specific tRNAs, and therefore to tailor effective aminoacyl-transfer catalysts. This method potentially provides a means of generating aminoacyl tRNAs that are charged with non-natural amino acids, which could be incorporated into proteins through cell-free translation.     

Lysophosphatidic acid,a growth factor-like lipid,in the saliva

Lysophosphatidic acid is a multifunctional phospholipid mediator and elicits a variety of biological responses in vitro and in vivo. Evidence is accumulating that lysophosphatidic acid plays important physiological roles in diverse mammalian tissues and cells. In the present study, we first examined whether lysophosphatidic acid is present in human saliva. We found that a significant amount of lysophosphatidic acid is present in the saliva (0.785 nmol/ml). The predominant fatty acyl moiety of lysophosphatidic acid was 18:1n-9 + n-7 followed by 18:0 and 16:0. A small amount of lysoplasmanic acid, an alkyl ether-linked analog of lysophosphatidic acid, was also detected in the saliva (0.104 nmol/ml). We found that physiologically relevant concentrations of lysophosphatidic acid induced accelerated growth of cells of mouth, pharynx, and esophagus origin in vitro. Lysophosphatidic acid also induced rapid increases in the intracellular free Ca2+ concentrations in these cells. We obtained evidence that lysophosphatidic acid receptor mRNAs are actually present in these cells. These results strongly suggest that lysophosphatidic acid is involved in wound healing in the upper digestive organs such as the mouth, pharynx, and esophagus.     

Aquaporin isoforms responsive to salt and water stresses and phytohormones in radish seedlings

Aquaporins in the plasma and vacuolar membranes play a key role in the intercellular and intracellular water transport in plants. First, we quantitated the absolute amounts for mRNAs of eight aquaporin isoforms in hypocotyls of radish seedlings. Then, we investigated the effects of salt and water stresses (150 mM NaCl, 300 mM mannitol and 20% polyethylene glycol) and phytohormones (gibberellic acid, abscisic acid and brassinolide) on the mRNA and protein levels of aquaporins in the plasma membrane (RsPIP1-1, 1-2, 1-3, 2-1, 2-2 and 2-3) and vacuolar membrane (RsTIP1-1 and 2-1). The mRNA and protein levels of RsTIP1-1, RsTIP2-1, RsPIP1-1, RsPIP1-2 and RsPIP1-3 were comparatively constant. In contrast, mannitol treatment altered the mRNA levels of RsPIP2-1, RsPIP2-2 and RsPIP2-3 in roots. Immunoblot analysis showed that the RsPIP2-1 protein level was increased by NaCl treatment and decreased by treatment with mannitol and polyethylene glycol. Gibberellic acid and abscisic acid suppressed the levels of mRNAs of RsPIP2-1, RsPIP2-2 and RsPIP2-3 and the protein level of RsPIP2-1 in roots. On the other hand, the protein levels of RsPIP1-group members and RsTIPs were scarcely changed by these phytohormones. In the case of hypocotyls and cotyledons, the mRNA and protein levels of eight isoforms were not markedly affected by any treatment. These results indicate that aquaporins in the root, especially the RsPIP2 group, may be a stress responsive type of aquaporin at least in the protein level.     

Stimulation of elongation growth and cell wall loosening in rice coleoptiles under microgravity conditions in space

We analyzed the growth rate and the cell wall properties of coleoptiles of rice seedlings grown at 23.6 degrees C for 68.5, 91.5 and 136 h during the Space Shuttle STS-95 mission. In space, elongation growth of coleoptiles was stimulated and the cell wall extensibility increased. Also, the levels of the cell wall polysaccharides per unit length of coleoptiles and the relative content of the high molecular mass matrix polysaccharides decreased in space. These differences in the cell wall polysaccharides could be involved in increasing the cell wall extensibility, leading to growth stimulation of rice coleoptiles in space.     

Cannabinoid receptors and their endogenous ligands

Delta9-Tetrahydrocannabinol, a major psychoactive component of marijuana, has been shown to interact with specific cannabinoid receptors, thereby eliciting a variety of pharmacological responses in experimental animals and human. In 1990, the gene encoding a cannabinoid receptor (CB1) was cloned. This prompted the search for endogenous ligands. In 1992, N-arachidonoylethanolamine (anandamide) was isolated from pig brain as an endogenous ligand, and in 1995, 2-arachidonoylglycerol was isolated from rat brain and canine gut as another endogenous ligand. Both anandamide and 2-arachidonoylglycerol exhibit various cannabimimetic activities. The results of structure-activity relationship experiments, however, revealed that 2-arachidonoylglycerol, but not anandamide, is the intrinsic natural ligand for the cannabinoid receptor. 2-Arachidonoylglycerol is a degradation product of inositol phospholipids that links the function of cannabinoid receptors with the enhanced inositol phospholipid turnover in stimulated tissues and cells. The possible physiological roles of cannabinoid receptors and 2-arachidonoylglycerol in various mammalian tissues such as those of the nervous system are discussed.     

Dual action of isoprenols from herbal medicines on both PPARgamma and PPARalpha in 3T3-L1 adipocytes and HepG2 hepatocytes

Several herbal medicines improve hyperlipidemia, diabetes and cardiovascular diseases. However, the molecular mechanism underlying this improvement has not yet been clarified. In this study, we found that several isoprenols, common components of herbal plants, activate human peroxisome proliferator-activated receptors (PPARs) as determined using the novel GAL4 ligand-binding domain chimera assay system with coactivator coexpression. Farnesol and geranylgeraniol that are typical isoprenols in herbs and fruits activated not only PPARgamma but also PPARalpha as determined using the chimera assay system. These compounds also activated full-length human PPARgamma and PPARalpha in CV1 cells. Moreover, these isoprenols upregulated the expression of some lipid metabolic target genes of PPARgamma and PPARalpha in 3T3-L1 adipocytes and HepG2 hepatocytes, respectively. These results suggest that herbal medicines containing isoprenols with dual action on both PPARgamma and PPARalpha can be of interest for the amelioration of lipid metabolic disorders associated with diabetes.     

Characterization of the repetitive sequences in a 200-kb region around the rice waxy locus: diversity of transposable elements and presence of veiled repetitive sequences

Repetitive genomic sequences might have various structural features and properties distinct from those of the known transposable elements (TE). Here, the content and properties of the repetitive sequences present in a 200-kb region around the rice waxy locus were analyzed using the available rice genomic database. In our previous Southern blotting analysis, 70% of the segments in this region showed smeared patterns, but according to the present database analysis, the proportion of repetitive sequences in this region was only 15%. The repetitive segments in this 200-kb region comprised 75 repetitive sequences that we classified into 46 subfamilies: 21 subfamilies were known TEs or repetitive sequences and 25 subfamilies consisted of newly identified TEs or novel types of repetitive sequences. The region contains no long terminal repeat (LTR) retrotransposable elements, but miniature inverted repeat transposable elements (MITEs) constituted a major class among the elements identified. These MITEs showed remarkable structural divergence: 12 elements were found to be new members of known MITE superfamilies, while five elements had novel terminal structures, and did not belong to any known TE families. Interestingly, about 10% of the repetitive sequences, including virus-like sequences did not have any of the usual characteristics of TEs, suggesting that a certain proportion of repetitive sequences that might not share the transpositional mechanisms of known elements are dispersed in the compact rice genome.     

Cyclooxygenase-2 inhibitor inhibits the fracture healing

We investigated the effects of cyclooxigenase-2 (cox-2) on fracture healing. After closed non-displaced fractures were created at the middle of both femoral shafts in 12-week-old Wister rats, a cox-2 specific inhibitor, etodolac (20 mg/day; intra-peritoneal) was administered every day for three weeks (E group). Bone union and callus formation were evaluated by weekly radiographs. Three weeks after surgery, the mechanical strength of the fractured femur was evaluated by a three-point-bending test. These results were compared with those of a vehicle control group (V group). The fracture healing score on radiographs in the E group three weeks after the surgery was 3.3 +/- 0.9, and in the V group it was 5.8 +/- 1.5, indicating that fracture healing was significantly poorer in the E than the V group (p < 0.05). From the three point bending test, the ultimate strength and stiffness of etodolac-treated fractured femurs were shown to be significantly lower than those in vehicle control group (p < 0.05). Mechanically, femurs of etodolac treated rats were weaker than those of control rats. Thus, it was concluded that etodolac, a cox-2 specific inhibitor, inhibited fracture healing.