Direct identification of a mutation in OsSh1 causing non-shattering in a rice (Oryza sativa L.) mutant cultivar using whole-genome resequencing.

Loss of seed shattering has been regarded as a key step during crop domestication. Mutagenesis contributes to the development of novel crop cultivars with a desired seed-shattering habit in a relatively short period of time, but also to uncovering the genetic architecture of seed shattering. 'Minamiyutaka', a non-shattering indica rice cultivar, was developed from the easy-shattering cultivar 'Moretsu' by mutation breeding via gamma-ray irradiation. In present study, we observed significant differences in shattering habit, breaking tensile strength, and abscission zone structure between 'Moretsu' and 'Minamiyutaka'. Whole-genome mutation analysis of 'Minamiyutaka' newly identified a 13-bp deletion causing defective splicing in exon 3 of the OsSh1 gene which has previously been referred to as a candidate for controlling seed shattering. Using CRISPR/Cas9 gene editing, we demonstrated that loss-of-function mutation in OsSh1 causes non-shattering in rice. Furthermore, gene expression analysis suggests that OsSh1 may function downstream of qSH1, a known key gene involved in abscission zone differentiation. Nucleotide diversity analysis of OsSh1 in wild rice accessions and cultivars revealed that OsSh1 has been under strong selection during rice domestication, and a missense mutation might have contributed to the reduction of seed shattering from the wild progenitors to cultivated rice.

The Selection of Gamma-Ray Irradiated Higher Yield Rice Mutants by Directed Evolution Method.

We have succeeded in selecting four higher yield mutants from five gamma-ray irradiated high-yielding Japanese rice varieties using a novel approach. A total of 464 M2 plants which had heavier total panicle weights per plant were first selected from 9801 irradiated M2 plants. Their higher yields were confirmed by yield trials conducted for three years with a six to ten-pairwise replicated plot design. FukuhibikiH6 and FukuhibikiH8 were selected from an irradiated high-yielding variety Fukuhibiki and showed 1.2% to 22.5% higher yield than their original significantly. YamadawaraH3 was selected from an irradiated high-yielding variety Yamadawara and its yield advantages were 2.7% to 3.9%. However, there was no difference in the genotypes of the 96 SNP (single nucleotide polymorphism) markers between the higher yield mutants and their respective original varieties. The differences in the measured phenotypical traits between each mutant and its original variety were not constant and the actual differences were marginal. Therefore, the higher yields of the selected mutants were likely to have been caused by physiological traits rather than phenotypical traits. The selection method used in this study is an application of the directed evolution method which has long been commonly used in the substantial improvements of microorganisms and their proteins.

Genome sequencing of ion-beam-induced mutants facilitates detection of candidate genes responsible for phenotypes of mutants in rice.

Ion beams are physical mutagens used for plant and microbe breeding that cause mutations via a mechanism distinct from those of chemical mutagens or gamma rays. We utilized whole-exome sequencing of rice DNA in order to understand the properties of ion beam-induced mutations in a genome-wide manner. DNA libraries were constructed from selected carbon-ion-beam-induced rice mutants by capturing with a custom probes covering 66.3 M bases of nearly all exons and miRNAs predicted in the genome. A total of 56 mutations, including 24 single nucleotide variations, 23 deletions, and 5 insertions, were detected in five mutant rice lines (two dwarf and three early-heading-date mutants). The mutations were distributed among all 12 chromosomes, and the average mutation frequency in the M1 generation was estimated to be 2.7 × 10-7 per base. Many single base insertions and deletions were associated with homopolymeric repeats, whereas larger deletions up to seven base pairs were observed at polynucleotide repeats in the DNA sequences of the mutation sites. Of the 56 mutations, six were classified as high-impact mutations that caused a frame shift or loss of exons. A gene that was functionally related to the phenotype of the mutant was disrupted by a high-impact mutation in four of the five lines tested, suggesting that whole-exome sequencing of ion-beam-irradiated mutants could facilitate the detection of candidate genes responsible for the mutant phenotypes.

Development of 'Darumadattan', a semidwarf lodging-resistant Tartary buckwheat cultivar, using gamma-ray irradiation.

We developed a new cultivar of Tartary buckwheat (Fagopyrum tataricum Gaertn.), 'Darumadattan'. This is the first semidwarf Tartary buckwheat cultivar to be developed by mutation breeding using gamma-ray irradiation. In 1999, 100 dry seeds of the leading Japanese cultivar, 'Hokkai T8' (known at that time as 'Hokkei 1'), were gamma-ray-irradiated with a total dose of 500 Gy (25 Gy/h × 20 h) at the Institute of Radiation Breeding (IRB), National Institute of Agrobiological Sciences, Hitachiomiya, Ibaraki, Japan. The seeds were sown in August 1999 in a field at IRB, and M2 seeds were collected from the eight individual plants that survived. In August 2000, 240 M2 seeds were sown in a field, and one semidwarf plant was found. The line named 'IRBFT-20' developed from the selected plant was investigated for its semidwarf characteristic and genetic stability in 2001-2005. 'IRBFT-20' was submitted for registration in 2011 and registered as the cultivar 'Darumadattan' in 2013. This name was chosen because the plants resemble "Daruma dolls" and "dattan" means "Tartary" in Japanese. 'Darumadattan' is a highly lodging-resistant and high-yielding cultivar and is expected to be used as both a commercial cultivar and a crossing parent.

Development of common buckwheat cultivars with high antioxidative activity -'Gamma no irodori', 'Cobalt no chikara' and 'Ruchiking'.

To breed new highly antioxidative common buckwheat cultivars, we selected individual plants from gamma ray-irradiated populations. Selection and propagation were repeated 4 or 5 times. This recurrent selection process resulted in many individuals with enhanced antioxidative activity. Among them, 2 individuals from the forth selection and 9 individuals from the fifth selection were developed into lines with increased antioxidative activities and diverse polyphenolic composition. From these lines, 2 new cultivars 'Gamma no irodori' and 'Cobalt no chikara' were developed. Furthermore, following the selection of individuals with high rutin contents, 'Ruchiking' was developed.

Comparison and Characterization of Mutations Induced by Gamma-Ray and Carbon-Ion Irradiation in Rice (Oryza sativa L.) Using Whole-Genome Resequencing.

Gamma-rays are the most widely used mutagenic radiation in plant mutation breeding, but detailed characteristics of mutated DNA sequences have not been clarified sufficiently. In contrast, newly introduced physical mutagens, e.g., heavy-ion beams, have attracted geneticists' and breeders' interest and many studies on their mutation efficiency and mutated DNA characteristics have been conducted. In this study, we characterized mutations induced by gamma rays and carbon(C)-ion beams in rice (Oryza sativa L.) mutant lines at M5 generation using whole-genome resequencing. On average, 57.0 single base substitutions (SBS), 17.7 deletions, and 5.9 insertions were detected in each gamma-ray-irradiated mutant, whereas 43.7 single SBS, 13.6 deletions, and 5.3 insertions were detected in each C-ion-irradiated mutant. The structural variation (SV) analysis detected 2.0 SVs (including large deletions or insertions, inversions, duplications, and reciprocal translocations) on average in each C-ion-irradiated mutant, while 0.6 SVs were detected on average in each gamma-ray-irradiated mutant. Furthermore, complex SVs presumably having at least two double-strand breaks (DSBs) were detected only in C-ion-irradiated mutants. In summary, gamma-ray irradiation tended to induce larger numbers of small mutations than C-ion irradiation, whereas complex SVs were considered to be the specific characteristics of the mutations induced by C-ion irradiation, which may be due to their different radiation properties. These results could contribute to the application of radiation mutagenesis to plant mutation breeding.

[The originality and creativity of leprosy sequelae patients in regard to dental care].

Leprosy sequelae patients surmount the inconvenience by the handicap. When people with the handicap are able to do the same act as healthy people, we may not pay attention to the originality and creativity for it. Therefore, we are not able to notice some risks which are hidden. We turned our attention to the denture in the oral region. The methods of putting in and taking out the denture of leprosy sequelae patients are characteristic. Even a partial denture, there are many patients who take out their denture using only their tongue, without touching a clasp with a finger. And they put in the denture by biting. But we had never not announced the details of their methods. So, we investigated how to handle their denture in National Sanatorium Nagashima Aiseien. We explain their methods and show the results. On the other hand, there are a lot of cases of the denture breakage in National Sanatorium Nagashima Aiseien. We think most of these matters occur due to their handicaps. We researched on the cases with dentures broken. In addition, we also describe our countermeasure.

[The characteristic of oral status and dental treatment of leprosy sequelae patients].

The leprosy sequelae especially the handicap of eyes and hands makes difficult to maintain the oral health status. So, leprosy sequelae patients are controlling themselves by creative and original methods. However even such efforts have hardly reached the level to stop of caries and periodontal disease. More support is required in order for them to maintain the oral health status. Therefore, in National Sanatorium Nagashima Aiseien, we started the periodic preventive system containing oral check-up, mouth cleaning, and early detection early treatment 15 years before. It is the report about this activity. First, the leprosy sequelae handicap which makes oral control difficult is described. Secondly, our periodic preventive system in National Sanatorium Nagashima Aiseien is explained. In order to evaluate this activity, the number of patients and the contents of dental treatment were compared. Furthermore, the number of remaining teeth was compared with the adult Japanese. Our periodic preventive system was received and they have many remaining teeth now. It is sure that this activity for 15 years was successful.

Gender-related differences in expression and function of hepatic P-glycoprotein and multidrug resistance-associated protein (Mrp2) in rats.

To clarify whether gender-related differences exist in the expression and function of hepatic P-glycoprotein- and/or multidrug resistance-associated protein (Mrp2), we measured the hepatobiliary excretion of doxorubicin and their protein levels in male and female Sprague-Dawley rats. When rats received a single intravenous injection of doxorubicin (5 mg/kg), a delay in the disappearance of doxorubicin from plasma was observed in male rats. When rats received a constant-rate infusion of doxorubicin, no significant gender-related differences in the apparent biliary clearance of doxorubicin based on the steady state plasma concentrations were observed between male and female rats. However, the net biliary clearance of doxorubicin based on the liver concentration, which represents the actual function of P-glycoprotein and/or Mrp2, was higher in female rats than in male rats. These results suggest that the actual function of the hepatobiliary transport of doxorubicin is greater in female than in male rats. Western blot analysis revealed that the expression of P-glycoprotein and Mrp2 in the liver of female rats was significantly higher than in male rats, similar to results of hepatobiliary excretion experiments. The expression of hepatic cytochrome P450 (CYP) 2B1, which is involved in the metabolism of doxorubicin, was significantly higher in male than in female rats. By pretreatment with testosterone (10 mg/day for 7 days), the actual biliary clearance of doxorubicin in female rats was nearly that of male rats. The protein levels of P-glycoprotein and Mrp2 in female rats were also lowered by treatment with testosterone so as to be nearer those in male rats. These results suggest that gender-related differences exist in P-glycoprotein- and Mrp2-mediated hepatobiliary transport and that these two transporters may be regulated by sex hormones.

Role of tumor necrosis factor-alpha in down-regulation of hepatic cytochrome P450 and P-glycoprotein by endotoxin.

We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in the down-regulation of hepatic P-glycoprotein and cytochrome P450 (CYP) by endotoxin, using TNF-alpha gene-deficient (TNF-alpha-/-) mice. In the case of P-glycoprotein, endotoxin (10 mg/kg) significantly decreased the expression of hepatic P-glycoprotein in wild-type mice 6 h, but not 24 h, after intraperitoneal injection, with no significant differences in the constitutional expression of P-glycoprotein between wild-type mice and TNF-alpha-/- mice. However, endotoxin had no effect on the expression of P-glycoprotein in TNF-alpha-/- mice either 6 or 24 h after injection. When doxorubicin was administered intravenously to TNF-alpha-/- mice treated 6 h earlier with and without endotoxin, no significant differences in the plasma concentrations of doxorubicin 3 h after injection were observed between endotoxin-treated and untreated TNF-alpha-/- mice. These results suggest that TNF-alpha plays a pivotal role in the down-regulation of P-glycoprotein by endotoxin. In the case of CYP, the constitutive expression of hepatic CYP3A2 and CYP2C11 had a tendency to decline in TNF-alpha-/- mice compared with that in wild-type mice. Endotoxin significantly decreased the expression of hepatic CYP3A2 and CYP2C11 in wild-type mice 24 h after injection, and that decreased expression was significantly greater in TNF-alpha-/- mice than wild-type mice. When antipyrine was administered intravenously to wild-type mice and TNF-alpha-/- mice treated 24 h earlier with endotoxin, the plasma concentrations of antipyrine in TNF-alpha-/- mice 3 h after injection were significantly higher than those in wild-type mice. These findings suggest that TNF-alpha plays a key role in endotoxin-induced down-regulation of hepatic P-glycoprotein, as well as plays a protective role in the regulation of hepatic CYP3A2 and CYP2C11 against endotoxin-induced acute inflammatory response. In TNF-alpha-/- mice, other cytokines appear to function as compensation for the lack of endogenous TNF-alpha.

Possible involvement of P-glycoprotein in renal excretion of pazufloxacin in rats.

The present study aims to investigate whether pazufloxacin, a new quinolone antimicrobial agent, is a substrate for P-glycoprotein in vitro, and whether it is excreted from kidney by P-glycoprotein and/or multidrug resistance-associated protein (Mrp2) in vivo. The in vitro experiments showed that the intracellular accumulation of pazufloxacin in adriamycin-resistant human chronic myelogenous leukemia cells (K562/ADR) overexpressing P-glycoprotein was significantly lower than that in human chronic myelogenous leukemia cells (K562/S) not expressing P-glycoprotein. When rats received an intravenous injection of pazufloxacin in combination with or without cyclosporine, cyclosporine significantly delayed the disappearance of pazufloxacin from plasma and decreased the systemic clearance and volume of distribution at steady state of pazufloxacin to 50% and 70% of the corresponding control values, respectively. Renal handling experiments revealed that the renal clearance of pazufloxacin was 75% of that corresponding to the systemic clearance, suggesting that the main route of pazufloxacin elimination is the kidney. Cyclosporine significantly increased the steady-state concentration of pazufloxacin in plasma by decreasing the tubular secretion clearance and glomerular filtration rate. These results suggest the possibility that pazufloxacin is excreted into the urine via P-glycoprotein. No significant differences in the renal and tubular secretion clearances of pazufloxacin were observed between normal rats and Eisai hyperbilirubinemic rats (EHBR), which have a hereditary deficiency in Mrp2, indicating the lack of the involvement of Mrp2 in the renal excretion of pazufloxacin. Sparfloxacin, a P-glycoprotein substrate, also significantly decreased the renal and tubular secretion clearances of pazufloxacin, suggesting that pazufloxacin and sparfloxacin share the same transporters, including P-glycoprotein. The present study at least suggests that pazufloxacin is excreted into the urine via P-glycoprotein and some active drug transporters other than Mrp2.

Analysis of information submitted by clinical trial sponsors regarding the safety of investigational drugs.

During performance of clinical trials in medical institutions, information regarding the safety of investigational drugs is submitted by trial sponsors according to guidelines for good clinical practice. In the present study, reports of clinical trials conducted at the University of Tokyo Hospital were examined, focusing on the safety information provided to the Institutional Review Board (IRB). Two hundred two reports (52 protocols) of safety information were submitted to the IRB by clinical trial sponsors between April 2000 and March 2001, of which 185 contained a total of 3021 cases of adverse events. Of those, 194 reports were judged by clinical investigators/physicians not to be associated with any significant problems and the trials were continued. For 157 of those 194 reports, it was considered unnecessary to inform the test subjects of the report contents, including the adverse events. The decision of whether or not the test subjects should be informed of such contents tended to depend on the causal relationship between the adverse events and drug intake, as well as the predictability of the adverse events. For 8 of those 194 reports, the IRB recommended that the clinical investigators/ physicians provide information to the test subjects and/or submit detailed information on the status of these subjects to the IRB. From these results, we suggest that establishment of a system to unify and evaluate drug safety information is necessary to provide safe and efficient clinical trials.

Azithromycin reverses anticancer drug resistance and modifies hepatobiliary excretion of doxorubicin in rats.

The present study aims to investigate whether azithromycin reverses P-glycoprotein-dependent anticancer drug resistance in vitro and modifies the hepatobiliary excretion of doxorubicin, a substrate for P-glycoprotein in vivo. Azithromycin increased dose-dependently the intracellular accumulation of doxorubicin in adriamycin-resistant human myelogenous leukemia cells (K562/ADR) with no effect on the expression of P-glycoprotein in the cells. However, the inhibitory effect was much weaker than that of cyclosporin A and was comparable to that of erythromycin. When Sprague-Dawley (SD) rats, which have drug transporting P-glycoprotein and multidrug resistance-associated protein 2 (Mrp2) in the bile canalicular membrane of hepatocytes, received an infusion of doxorubicin, the steady-state biliary clearance of doxorubicin was significantly decreased for 40 min after a single intravenous injection of azithromycin. However, azithromycin did not increase the plasma concentration of doxorubicin. The biliary clearance of doxorubicin in Eisai hyperbilirubinemic rats (EHBRs), which have a hereditary deficiency in Mrp2, was significantly decreased compared with that in Sprague-Dawley rats, suggesting the involvement of Mrp2 in the biliary excretion of doxorubicin. The present findings suggest that azithromycin overcomes P-glycoprotein-dependent anticancer drug resistance of tumors by inhibiting the binding of doxorubicin to P-glycoprotein in K562/ADR cells and inhibits the hepatobiliary excretion of drugs that are substrates for P-glycoprotein and Mrp2.

Periodontal disease and diabetes mellitus: the role of tumor necrosis factor-alpha in a 2-way relationship.

It is generally accepted that obesity is associated with many other multiple-risk factor syndromes such as hypertension, hyperlipidemia, type 2 diabetes mellitus, and periodontal disease. The number of obese people is increasing rapidly in both western and eastern countries. Adipocytes in the adipose tissues of obese people produce large quantities of biologically active molecules such as leptin, an important molecule regulating energy expenditure and body weight. Therefore, adipocyte-derived active molecules, named adipocytokines, are candidate molecules accounting for the close association between obesity and other multiple-risk factor syndromes. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is produced by adipocytes, and its blood concentration is elevated in obese patients and declines with weight loss. Studies have demonstrated that TNF-alpha suppresses insulin action via its specific receptor; hence, it exacerbates insulin resistance. In addition to adipocytes, monocytes/macrophages produce large quantities of TNF-alpha. Thus, TNF-alpha, produced from monocytic cells due to inflammatory diseases, may have an additive influence on insulin sensitivity to adipocyte-derived TNF-alpha. Here, we hypothesized that 1) TNF-alpha produced by the adipose tissues of obese patients acts as a risk factor for periodontal inflammation, and 2) TNF-alpha produced due to periodontal inflammation may be an additional important factor influencing insulin sensitivity in both obese and type 2 diabetic patients. We believe that this interaction is a possible mechanism accounting for a 2-way relationship between type 2 diabetes and periodontal disease.