Usefulness of 11C-methionine-PET for predicting the efficacy of carbon ion radiation therapy for head and neck mucosal malignant melanoma.

The aim of this study was to determine whether l-methyl-[11C]-methionine (MET) positron emission tomography (PET) allows the prediction of outcomes in patients with head and neck mucosal malignant melanoma treated with carbon ion radiation therapy (CIRT). This was a retrospective cohort study involving 85 patients who underwent a MET-PET or MET-PET/computed tomography (CT) examination before and after CIRT. MET uptake in the tumour was evaluated semi-quantitatively using the tumour-to-normal tissue ratio (TNR). Local recurrence, metastasis, and outcome predictions were studied in terms of TNR before CIRT (TNRpre), TNR after CIRT (TNRpost), and the TNR change ratio. Kaplan-Meier curves revealed significant differences between patients with higher TNRpre values and those with lower TNRpre values in regard to local recurrence, metastasis, and outcome (log-rank test P<0.0001 for all three). There were also significant differences in metastasis rates and outcomes between patients with higher and lower TNRpost values (log-rank test P=0.0105 and P=0.027, respectively). The Cox proportional hazards model revealed TNRpre to be a factor significantly influencing the risk of local recurrence (hazard ratio (HR) 29.0, P<0.001), risk of metastasis (HR 2.67, P=0.024), and the outcome (HR 6.3, P<0.001). MET-PET or MET-PET/CT is useful for predicting the outcomes of patients with head and neck mucosal malignant melanoma treated with CIRT.

A pilot study of evaluation of the antioxidative activity of resveratrol and its analogue in a 6-month feeding test in young adult mice.

Resveratrol, a polyphenolic phytoalexin, has free-radical scavenging activity and we found that it induces chromosomal aberrations, micronuclei, and sister chromatid exchanges in vitro. We synthesized its analogue 4-hydroxy-trans-stilbene (4-OH) and found that it has the same in vitro clastogenic activities as resveratrol, suggesting that the 4' hydroxy group of resveratrol is responsible for the effect. We fed resveratrol and 4-OH to young adult ICR mice at 0, 0.2, 2, or 20 ppm in their standard powder diet for 6 months and investigated the antioxidative effects. Half of each group was given 3000 ppm potassium bromate (KBrO(3)) in water for the last week to cause oxidative damage. Body weight gain tended to increase in males at 0.2 ppm resveratrol or 4-OH, and in females at 2 ppm 4-OH. Micronucleus (MN) analysis in bone marrow erythrocytes showed that the KBrO(3) tendency to induce MN was not prevented by the dietary resveratrol or 4-OH, which themselves did not induce MN under the present conditions. In this pilot study, resveratrol and 4-OH showed no obvious effect, either beneficial or adverse, at doses that are feasible in daily life for humans.

Isolation of mutant lines with decreased numbers of chloroplasts per cell from a tagged mutant library of the moss Physcomitrella patens.

Eleven mutant lines exhibiting decreased numbers of chloroplasts per cell were isolated from 8 800 tagged mutant lines of Physcomitrella patens by microscopic observations. Chloronema subapical cells in wild-type plants had a mean of 48 chloroplasts, whereas chloroplast numbers in subapical cells in mutant lines 215 and 222 decreased to 75 % of that in the wild type. Seven mutant lines - 473, 122, 221, 129, 492, 207, and 138 - had about half as many chloroplasts as the wild type. Mutant line 11 had a few remarkably enlarged chloroplasts, and mutant line 347 had chloroplasts of various sizes. Whereas the cell volume was the same as in the wild type in mutant lines 222, 473, 221, 129, 492, and 207, the cell volume of the other mutants increased. The chloroplast number of leaf cells was the same as that of chloronema cells in each mutant line when gametophores could be formed. Treatment with ampicillin decreased the number of chloroplasts in all mutant lines. Southern hybridization using DNA in tags as probes showed that only one insertion occurred in mutant lines 473 and 221. To determine whether the tagged DNA inserted into the known genes for plastid division, we isolated the PpMinD1, PpMinD2, and PpMinE1 genes. Genomic polymerase chain reaction analysis showed that the PpFtsZ and PpMinD/E genes were not disrupted by the insertion of the tags in mutant lines 11 and 347, respectively.

Biosystematic studies on the family Tofieldiaceae I. Phylogeny and circumscription of the family inferred from DNA sequences of matK and rbcL.

In order to specify phylogenetic positions of the genera of Tofieldiaceae (Tofieldia, Triantha, Pleea, Harperocallis, Isidrogalvia), and to suggest reasonable circumscription of the family and genera of Tofieldiaceae, we determined DNA sequences of matK and rbcL for each genus of the family, and analyzed them phylogenetically with the 45 families and 113 genera of the monocots other than Tofieldiaceae, whose matK and rbcL sequences have already been reported. We found that Tofieldia, Triantha, Pleea, and Harperocallis form the same clade, which receives 100% bootstrap support. This clade can be regarded as corresponding to Tofieldiaceae, and is embedded in the clade of Alismatales (98%). On the other hand, Isidrogalvia is not included in this Tofieldiaceae clade, and positioned as sister to Narthecium (100%), embedded in the clade of Nartheciaceae (Dioscoreales) (100%). In the Tofieldiaceae, Pleea first diverges from the remaining three genera (100%), and then, Harperocallis diverges from the Tofieldia- Triantha complex (100%). In the Tofieldia- Triantha complex, five Tofieldia species form the same clade (100%), and two Triantha species form another clade (100%). Thus, Isidrogalvia should be transferred from Tofieldiaceae to Nartheciaceae. As Isidrogalvia, as well as the Nartheciaceae, have the carpels that are fully connate into a single style, Isidrogalvia fits the Nartheciaceae well with respect to carpel connation. After this transfer, the Tofieldiaceae correspond mainly to plants with almost free carpels and three styles. Pleea is better treated as an independent genus than included in Tofieldia. Triantha can be treated either as an independent genus or as congeneric with Tofieldia.

Survival of Tdc transposable elements of the En/Spm superfamily in the carrot genome.

Three subfamilies of the En/Spm-type transposable element of carrot, Tdc A, B, and C, were characterized. It was supposed that the Tdc A subfamily may include autonomous elements which can produce transposases. Tdc B elements are defective, but still generate transcripts containing mutant open reading frame (ORF) sequences for transposases. The single member of the Tdc C group recovered seems to be a pseudogene. The sequences of the transposase ORFs of Tdc A and Tdc B elements are more highly conserved than those of the 5; and 3; untranslated regions and introns, as is found in other structural genes that are subject to selection. These observations indicate that the mutations in the nucleotide sequences of the Tdc elements occurred in the host genome. However, the mutations in the 5; and 3; untranslated regions and introns, which may not be sufficient to prevent transposition, accumulated in autonomous elements, which could transpose and produce copies. When the reproduction rate and the rate of disabling mutations reached an equilibrium, that is, when the birth rate of the transposable elements in the genome equalled the death rate, the population of elements achieved a stationary state in the genome, and could thus survive.

Establishment of gene-trap and enhancer-trap systems in the moss Physcomitrella patens.

Because of its simple body plan and ease of gene knockout and allele replacement, the moss Physcomitrella patens is often used as a model system for studies in plant physiology and developmental biology. Gene-trap and enhancer-trap systems are useful techniques for cloning genes and enhancers that function in specific tissues or cells. Additionally, these systems are convenient for obtaining molecular markers specific for certain developmental processes. Elements for gene-trap and enhancer-trap systems were constructed using the uidA reporter gene with either a splice acceptor or a minimal promoter. Through a high rate of transformation conferred by a method utilizing homologous recombination, 235 gene-trap and 1073 enhancer-trap lines were obtained from 5637 and 3726 transgenic lines, respectively. The expression patterns of these trap lines in the moss gametophyte varied. The candidate gene trapped in a gene-trap line YH209, which shows rhizoid-specific expression, was obtained by 5' and 3' RACE. This gene was named PpGLU, and forms a clade with plant acidic alpha-glucosidase genes. Thus, these gene-trap and enhancer-trap systems should prove useful to identify tissue- and cell-specific genes in Physcomitrella.

Evolution of MADS-box gene induction by FLO/LFY genes.

Some MADS-box genes function as floral homeotic genes. The Arabidopsis LFY gene is a positive regulator of floral homeotic genes, and homologs of the FLO/LFY gene family in other angiosperms and gymnosperms are likely to have a similar function. To investigate the origin of the floral homeotic gene regulatory cascade involving the FLO/LFY gene, FLO/LFY homologs were cloned from a leptosporangiate fern (Ceratopteris richardii), two eusporangiate ferns (Angiopteris lygodiifolia and Botrychium multifidum var. robustum), three fern allies (Psilotum nudum, Equisetum arvense, and Isoetes asiatica), and a moss (Physcomitrella patens). The FLO/LFY gene phylogenetic tree indicates that both duplication and loss of FLO/LFY homologs occurred during the course of vascular plant evolution. The expression patterns of the Ceratopteris LFY genes (CrLFY1 and 2) were assessed. CrLFY1 expression was prominent in tissues including shoot tips and circinate reproductive leaves, but very weak in other tissues examined. Expression of CrLFY2 was also prominent in tissues, including shoot tips and circinate reproductive leaves. These patterns of expression are dissimilar to that of any Ceratopteris MADS-box gene previously reported, suggesting that the induction of MADS-box genes by FLO/LFY is not established at the stage of ferns.

Isolation of homeodomain-leucine zipper genes from the moss Physcomitrella patens and the evolution of homeodomain-leucine zipper genes in land plants.

Homeobox genes encode transcription factors involved in many aspects of developmental processes. The homeodomain-leucine zipper (HD-Zip) genes, which are characterized by the presence of both a homeodomain and a leucine zipper motif, form a clade within the homeobox superfamily and were previously reported only from vascular plants. Here we report the isolation of 10 HD-Zip genes (named PPHB:1-PPHB:10) from the moss Physcomitrella patens. Based on a phylogenetic analysis of the 10 PPHB: genes and previously reported vascular plant HD-Zip genes, all of the PPHB: genes except Pphb3 belong to three of the four HD-Zip subfamilies (HD-Zip I, II, and III), indicating that these subfamilies originated before the divergence of the vascular plant and moss lineages. Pphb3 is sister to the HD-Zip II subfamily and has some distinctive characteristics, including the difference of the a(1) and d(1) sites of its leucine zipper motif, which are well conserved in each HD-Zip subfamily. Comparison of the genetic divergence of representative HD-Zip I and II genes showed that the evolutionary rate of HD-Zip I genes was faster than that of HD-Zip II genes.

Phylogeny of the lady fern group, tribe Physematieae (Dryopteridaceae), based on chloroplast rbcL gene sequences.

Nucleotide sequences of the chloroplast gene rbcL from 42 species of the fern tribe Physematieae (Dryopteridaceae) were analyzed to gain insights into the inter- and intrageneric relationships and the generic circumscriptions in the tribe. The phylogenetic relationships were inferred using the neighbor-joining and maximum-parsimony methods, and both methods produced largely congruent trees. These trees reveal that: (1) Athyrium, Cornopteris, Pseudocystopteris, and Anisocampium form a clade and Athyrium is polyphyletic; (2) Deparia sensu lato is monophyletic and Dictyodroma formosana is included in the Deparia clade; (3) Diplaziopsis forms a clade with Homalosorus, which is isolated from the other genera of the Physematieae; (4) Monomelangium is included in the monophyletic Diplazium clade; and (5) Rhachidosorus is not closely related to either Athyrium or Diplazium.

Prevention of N-methylnitrosourea-induced colon carcinogenesis in rats by oxygenated carotenoid capsanthin and capsanthin-rich paprika juice.

Epidemiological and animal studies have provided evidence that dietary carotenoids may reduce the risk of certain types of cancer. An inhibitory activity of oxygenated carotenoid capsanthin, a potent antioxidant, and paprika juice rich in capsanthin (3.54 mg/100 ml) against colon carcinogenesis was investigated in F344 rats. In Experiment I (short-term assay), six rats each were given a gavage of 5 mg, 0.2 mg, or 0.008 mg capsanthin six times a week for Weeks 2-6 after receiving three intrarectal doses of 4 mg N-methylnitrosourea in Week 1. The number of colonic aberrant crypt foci, preneoplastic lesions, at Week 6 was significantly fewer (by 42%) in the 0.2 mg capsanthin group, but not in other groups, than the control group. In Experiment II (long-term assay), five groups of 30 or 25 rats each received an intrarectal dose of 2 mg N-methylnitrosourea three times a week for Weeks 1-3, and had either of 10 p.p.m. or 2 p.p.m. capsanthin solutions, 1:2.5 and 1:16.7 diluted solution of paprika juice (containing 10 p.p.m. or 2 p.p.m. capsanthin), and tap water (control fluid) as drinking fluid throughout the experiment. The experimental groups were fed 0.2 mg or 0.04 mg capsanthin/day/rat. The colon cancer incidence at Week 30 was significantly lower in the highly diluted paprika juice group (40%), but not in the moderately diluted paprika juice group (60%) and the capsanthin solution groups (68% and 68%) than the control group (83%). The results suggested that paprika juice may affect colon carcinogenesis. However, capsanthin alone failed to inhibit colon tumorigenesis, in spite of suppression of aberrant crypt foci formation in the short-term assay. Further studies are needed to explain this discrepancy.

Tagged mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle mutagenesis.

The moss, Physcomitrella patens has been used as a useful material in many fields, because of its simple body plan, ease of gene targeting, and other reasons. Although many mutants have been reported, no method to isolate the corresponding genes was reported. We developed a gene tagging and gene-trap system in P. patens by using the shuttle mutagenesis technique, which has been used in the budding yeast. In 5264 tagged lines, 203 mutants with altered developmental or morphological phenotypes were obtained. In 129 of 4757 gene-trap lines, beta-glucuronidase (GUS) activity was detected in some tissue. Although multiple copies of a tag were detected in many tagged lines by Southern analyses, most copies are likely integrated at the same locus according to PCR analyses.

Molecular phylogeny of Coriaria, with special emphasis on the disjunct distribution.

Coriaria, which has the most conspicuously disjunct distribution of the flowering plants, is distributed in four separate areas of the world. The phylogenetic relationships of 12 Coriaria species collected from the representative disjunct areas were inferred by comparing 2416 bp of the combined data set of rbcL (a large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase) and matK (maturase K) genes. The phylogenetic tree shows that the Chile-Papua New Guinea-New Zealand-Pacific islands species and the Central America-northern South America species form a sister group, and the Eurasian clade is more basal to them. The divergence time between the Eurasian group and the other species was estimated as 63 or 59 million years ago using rbcL and matK molecular clocks, respectively. These results do not support previously proposed hypotheses which explain the disjunct distribution on the basis of continental drift but suggest that the distribution pattern was formed by several geographical migrations and separations in the Cenozoic.