Boron toxicity in rice (Oryza sativa L.). I. Quantitative trait locus (QTL) analysis of tolerance to boron toxicity.

Boron toxicity tolerance of rice plants was studied. Modern japonica subspecies such as Koshihikari, Nipponbare, and Sasanishiki were tolerant, whereas indica subspecies such as Kasalath and IR36 were intolerant to excessive application of boron (B), even though their shoot B contents under B toxicity were not significantly different. Recombinant inbred lines (RILs) of japonica Nekken-1 and indica IR36 were used for quantitative trait locus (QTL) analysis to identify the gene responsible for B toxicity tolerance. A major QTL that could explain 45% of the phenotypic variation was detected in chromosome 4. The QTL was confirmed using a population derived from a recombinant inbred line which is heterogenic at the QTL region. The QTL was also confirmed in other chromosome segment substitution lines (CSSLs).

Distribution, structure, organ-specific expression, and phylogenic analysis of the pathogenesis-related protein-3 chitinase gene family in rice (Oryza sativa L.).

Rice (Oryza sativa L.) pathogenesis-related (PR)-3 chitinases, like other PR proteins, are each coded by one of the genes of a multigene family in the plant genome. We assembled the database information about rice PR-3 chitinase sequences. A total of 12 PR-3 chitinase loci (Cht1 to Cht12) were found deployed in the rice genome. Some of the loci were occupied by 2 or more alleles. For all the loci expect Cht4, Cht5, Cht6, and Cht11, the amino acid sequence was polymorphic between japonica and indica varieties of rice, but glutamic acid acting as a catalytic residue was completely conserved in all the loci expect Cht7. All the genes except Cht7, which was not tested in this study, were transcripted in some organs (leaf, sheath, root, and meristem) of rice plants. These results suggest that chitinase proteins encoded by the genes at these loci have important biological effects, at least antifungal activities, on rice plants. We also proposed a new classification of rice PR-3 chitinases based on their domain structures. This classification was consistent with the results of phylogenetic analysis of rice chitinases.

Identification of YAC clones containing the mutable slender glume locus slg in rice (Oryza sativa L.).

A mutable slender glume gene slg, which often reverts to the wild-type state, was induced by gamma-ray irradiation of seeds of the japonica rice cultivar 'Gimbozu'. The final goal was to understand whether the slender glume mutation was associated with the insertion of a transposable element, utilizing map-based cloning techniques. The RFLP (restriction fragment length polymorphism) analysis revealed that the slg locus was located between two RFLP loci, XNpb33 and R1440, on chromosome 7 with recombination values of 3.1% and 1.0%, respectively. Using these two RFLP loci as probes, five YAC (yeast artificial chromosome) clones containing either of these two loci were selected from a YAC library. Subsequently, both end fragments of these YAC clones, amplified by the inverse PCR (IPCR) method, were used to select new YAC clones more closely located to the slg locus. After repeating such a procedure, we successfully constructed a 6-cM YAC contig, and identified four overlapping YAC clones, Y1774, Y3356, Y5124, and Y5762, covering the slg locus. The chromosomal location of the slg was narrowed down to the region with a physical distance of less than 280 kb between the right-end fragments of Y1774 and Y3356.

Development of a novel functional biosensor with a short Ca(2+)-dependent deoxyribozyme.

We develop a novel functional biosensor on a deoxyribozyme. A 5'-end-immobilized short Ca(2+)-dependent deoxyribozyme (dCGCTGGCAGGCTACAACGAGTCTTC) binds to a target RNA substrate (rGAAGACA decrease UGCCAGCG; decrease denotes an RNA cleavage site), and acts as an enzyme in the presence of Ca2+. It cleaves the target RNA substrate at one site of rAp decrease U in the asymmetric internal loop.

Effects of metal ions and catalytic loop sequences on the complex formation of a deoxyribozyme and its RNA substrate.

A deoxyribozyme is a catalytic DNA that catalyzes a site-specific RNA cleavage activity and requires various divalent cations. Earlier we have reported that by downsizing the catalytic loop of a deoxyribozyme from 15-mer to 11-mer it resulted in a short and novel Ca2+-dependent deoxyribozyme. In this paper, we investigate the complex formation of deoxyribozymes with their RNA substrates by using surface plasmon resonance (SPR) in order to determine quantitatively the effect of Ca2+ or Mg2+ on the recognition step between a deoxyribozyme and its RNA substrate. The results indicate that both the association and dissociation rate constants (k(a) and k(d)) for the deoxyribozyme-RNA complex depends on metal ions as well as the loop size of the deoxyribozyme. Metal ions with high RNA cleavage activity induced an increase in k(a) and a decrease in k(d). On the basis of the results, we propose that Ca2+ ions may play a role in the rearrangement of the 11-mer catalytic loop of the short Ca2+-dependent deoxyribozyme.

Identification of mutable slender glume gene in rice (Oryza sativa L.).

The segregation pattern and chromosomal location of a slender glume mutation, induced by gamma-ray irradiation, was investigated. The mutation is genetically unstable: in the selfed progenies of slender glumed plants, not only plants with normal glumes but also plants that are chimeric for glume shape almost always appear at low frequency. The results showed that the mutation is controlled by a single recessive, mutable mutant gene slg. The frequency of reversion of slg to its wild-type state was little affected by crossing, back-crossing, genetic background or cytoplasmic factors. Conventional trisomic and linkage analyses revealed that the slg locus was located close to the rfs (rolled fine stripe leaf) locus on chromosome 7. In a subsequent RFLP analysis, slg was found to be located between the two RFLP loci XNpb20 and XNpb33, with recombination values of 3.0 and 3.2%, respectively. Southern analysis indicated that the mutability of slg is caused by none of the known transposable elements in rice. From these results, we infer that slg has a novel transposable DNA insert in its vicinity, which was possibly activated by gamma-ray irradiation.

Development of a short Ca2+-dependent deoxyribozyme with RNA cleavage activity.

We developed a short Ca2+-dependent deoxyribozyme with 11 mer catalytic loop domain (dGGCTACAACGA) that catalyzed site-specific RNA cleavage reaction between rA and rU. The second-order rate constant of this short deoxyribozyme is 1.7 x 10(7) M(-1) min(-1) at 37 degrees C, and this value is very similar to that of the deoxyribozyme (dGGCTAGCTACAACGA) in the presence of Ca2+.

Effect of substrate RNA sequence on the cleavage reaction by a short ribozyme.

Leadzyme is a ribozyme that requires Pb2+. The catalytic sequence, CUGGGAGUCC, binds to an RNA substrate, GGACC downward arrowGAGCCAG, cleaving the RNA substrate at one site. We have investigated the effect of the substrate sequence on the cleavage activity of leadzyme using mutant substrates in order to structurally understand the RNA catalysis. The results showed that leadzyme acted as a catalyst for single site cleavage of a C5 deletion mutant substrate, GGAC downward arrowGAGCCAG, as well as the wild-type substrate. However, a mutant substrate GGACCGACCAG, which had G8 deleted from the wild-type substrate, was not cleaved. Kinetic studies by surface plasmon resonance indicated that the difference between active and inactive structures reflected the slow association and dissociation rate constants of complex formation induced by Pb2+rather than differences in complex stability. CD spectra showed that the active form of the substrate-leadzyme complex was rearranged by Pb2+binding. The G8 of the wild-type substrate, which was absent in the inactive complex, is not near the cleavage site. Thus, these results show that the active substrate-leadzyme complex has a Pb2+binding site at the junction between the unpaired region (asymmetric internal loop) and the stem region, which is distal to the cleavage site. Pb2+may play a role in rearranging the bases in the asymmetric internal loop to the correct position for catalysis.

Sleep improvement by light in a demented aged individual.

We administered daily morning bright light exposure in two separate periods to a demented aged subject in a nursing home and analyzed the sleep pattern changes from the sleep diary over 6 months. In the first light exposure period, the ratio of sleep time in night hours increased and maintained a higher value throughout the period than the previous baseline. After stopping the light exposure, the sleep pattern of the subject deteriorated. Resuming the light exposure reproduced similar sleep pattern changes. These results suggest that morning bright light exposure provides a better environment for aged persons to maintain a regular sleep-wake pattern.

Efficient callus induction and plant regeneration from filaments with anther in lily (Lilium longiflorum Thunb.).

Bulb scale propagation makes it difficult to obtain a large number of bulblets from disease-free stocks in a short time. The establishment of improved micropropagation procedures by in vitro culture is therefore desirable. Easter lily (Lilium longiflorum Thunb.) filaments with and without anther were excised and cultured in vitro with different media and culture conditions. In cultures of filaments with anther, callus developed and led to bulb, shoot, and root formation, whereas in cultures of filaments lacking anther, callus development did not occur. Among the various media tested, the B5 medium combined with darkness and the N6 medium combined with darkness or light, both supplemented with 9% sucrose, proved to be superior. A total of 1260 plants were regenerated from callus, acclimatized under a mist, and transferred to the greenhouse with a 100% success rate. No morphological abnormalities were observed among plants regenerated from filament-derived callus and all plants displayed isozyme banding patterns identical to the original cultivar. Chromosome observations revealed that all callus-regenerated plantlets tested were diploid (2n=24). The results suggest that in vitro culture of filaments with anther can be cultured for mass propagation.

Penetration of imipenem and other antibiotics into inflammatory exudate and their efficacy in pseudomonas infection in the rat granuloma pouch model.

The rat granuloma pouch model was used to study the penetration of imipenem into inflammatory exudate and its efficacy in an experimental local infection. Rats were given a single intravenous injection of drug via the tail vein at a dose of 40 mg/kg. The peak concentrations of imipenem, ceftizoxime, cefazolin, and cefsulodin in exudates ranged from 10.1 to 12.3 mg/l, except that ampicillin achieved only 5.4 mg/l. Imipenem rapidly reached peak concentration after injection, but the half life for elimination of imipenem from exudate was 77.7 min, which was shorter than that of other beta-lactam antibiotics. Imipenem exhibited more rapid reduction in bacterial growth than cefsulodin in an experimental local infection with Pseudomonas aeruginosa in the pouch model. The results suggested that imipenem was an effective agent in treating the local infection.

Therapeutic effects of imipenem-cilastatin on experimental intrauterine infections in rats.

The therapeutic effects of imipenem-cilastatin (MK-0787-MK-791) on experimental intrauterine infections in progesterone-treated virgin rats and postpartum rats were studied. The relative efficacy of imipenem-cilastatin for the treatment of such intrauterine infections was compared with that of cefazolin and ampicillin for the treatment of infections caused by Escherichia coli and Streptococcus faecalis, respectively. Treatment with imipenem-cilastatin significantly inhibited the proliferation of E. coli and S. faecalis in uteri, as compared with the proliferation in untreated controls. Cefazolin failed to affect the E. coli infection. With the S. faecalis infection, ampicillin effectively reduced bacterial growth, as compared with that in untreated controls. However, ampicillin was inferior to and comparable to imipenem-cilastatin in progesterone-treated virgin rats and postpartum rats, respectively. A further experiment with S. faecalis infections in rats made neutropenic by intraperitoneal injection of cyclophosphamide showed that the therapeutic effectiveness of imipenem-cilastatin was superior to that of ampicillin and was not influenced by neutropenia. Our results suggest that imipenem-cilastatin may be a useful agent for the treatment of obstetric and gynecologic infections.