Asymbiotic mass production of the arbuscular mycorrhizal fungus Rhizophagus clarus.

Arbuscular mycorrhizal (AM) symbiosis is a mutually beneficial interaction between fungi and land plants and promotes global phosphate cycling in terrestrial ecosystems. AM fungi are recognised as obligate symbionts that require root colonisation to complete a life cycle involving the production of propagules, asexual spores. Recently, it has been shown that Rhizophagus irregularis can produce infection-competent secondary spores asymbiotically by adding a fatty acid, palmitoleic acid. Furthermore, asymbiotic growth can be supported using myristate as a carbon and energy source for their asymbiotic growth to increase fungal biomass. However, the spore production and the ability of these spores to colonise host roots were still limited compared to the co-culture of the fungus with plant roots. Here we show that a combination of two plant hormones, strigolactone and jasmonate, induces the production of a large number of infection-competent spores in asymbiotic cultures of Rhizophagus clarus HR1 in the presence of myristate and organic nitrogen. Inoculation of asymbiotically-generated spores promoted the growth of host plants, as observed for spores produced by symbiotic culture system. Our findings provide a foundation for the elucidation of hormonal control of the fungal life cycle and the development of inoculum production schemes.

Brain activity in the prefrontal cortex during cancelation tasks: Effects of the stimulus array.

Cancelation tasks have been widely used to neurologically assess selective attention and visual search in various clinical and research settings. However, there is still a lack of evidence regarding the effect of differences in array conditions on brain activity in the prefrontal cortex (PFC) and its association with developmental characteristics. This study employed cancelation tasks to investigate the effects of varying array conditions on oxygenated hemoglobin (oxy-Hb) concentrations. Data from 24 healthy adults were analyzed based on performance during two-block-design type of cancelation tasks with different array conditions (i.e., structured array vs. random array). Performance was assessed based on the number of correct responses, incorrect responses, hit ratios, and performance scores (PS); while PFC activity was examined using near-infrared spectroscopy. In addition, characteristics of attention-deficit/hyperactivity disorder (ADHD) were assessed using the ADHD-Rating Scale-IV (ADHD-RS-IV). Results revealed that the numbers of correct responses and PS were higher in the random array, but there was no difference in the incorrect responses and hit ratio. Similarly, we observed that the oxy-Hb concentration in the PFC significantly increased during the task. Additionally, in the structured array, a significant relationship between task performance and characteristics of ADHD was found but not in the random array. Our results regarding the above-mentioned changes in oxy-Hb concentration suggest that the PFC region is involved in selective attention. We also found that cancelation tasks in a structured array may be useful in evaluating the characteristics of ADHD.

The effects of ERN1 on gene expression during early rhizobial infection in Lotus japonicus.

Legumes develop root nodules in association with compatible rhizobia to overcome nitrogen deficiency. Rhizobia enter the host legume, mainly through infection threads, and induce nodule primordium formation in the root cortex. Multiple transcription factors have been identified to be involved in the regulation of the establishment of root nodule symbiosis, including ERF Required for Nodulation1 (ERN1). ERN1 is involved in a transcription network with CYCLOPS and NODULE INCEPTION (NIN). Mutation of ERN1 often results in misshapen root hair tips, deficient infection thread formation, and immature root nodules. ERN1 directly activates the expression of ENOD11 in Medicago truncatula to assist cell wall remodeling and Epr3 in Lotus japonicus to distinguish rhizobial exopolysaccharide signals. However, aside from these two genes, it remains unclear which genes are regulated by LjERN1 or what role LjERN1 plays during root nodule symbiosis. Thus, we conducted RNA sequencing to compare the gene expression profiles of wild-type L. japonicus and Ljern1-6 mutants. In total, 234 differentially expressed genes were identified as candidate LjERN1 target genes. These genes were found to be associated with cell wall remodeling, signal transduction, phytohormone metabolism, and transcription regulation, suggesting that LjERN1 is involved in multiple processes during the early stages of the establishment of root nodule symbiosis. Many of these candidate genes including RINRK1 showed decreased expression levels in Ljnin-2 mutants based on a search of a public database, suggesting that LjERN1 and LjNIN coordinately regulate gene expression. Our data extend the current understanding of the pleiotropic role of LjERN1 in root nodule symbiosis.

Brain activity in the prefrontal cortex during a cancellation task: effects of the target-to-distractor ratio.

Cancellation tasks have been widely used to neurologically assess selective attention and visual search in various clinical and research settings. However, there is still a lack of evidence regarding the effect of the level of task difficulty on brain activity in the prefrontal cortex (PFC). This study implemented cancellation tasks to investigate the effects of varying task difficulty on oxygenated hemoglobin (oxy-Hb) concentrations. Data from 21 healthy adults were analyzed based on performance during three-block-design types of cancellation tasks with different T/D ratios (i.e., 1/9, 2/8, and 3/7). Performance was assessed via the number of correct responses, incorrect responses, hit ratios, achievement ratios, and performance scores (PS), while PFC activity was examined using near-infrared spectroscopy. Both the numbers of correct responses and PS were the lowest for the smallest T/D ratio. Similarly, we observed that the oxy-Hb concentration in the PFC was significantly increased during the task. Our results support the findings of previous studies that used conventional cancellation tasks, thus suggesting that block design types are suitable for examinations in the same contexts. Regarding the above-mentioned changes in the oxy-Hb concentration, the findings suggest that the PFC region is involved in selective attention.

Amlexanox enhances the antitumor effect of anti-PD-1 antibody.

Cancer immunotherapy, especially treatment with monoclonal antibodies (mAbs) that block programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) signaling, has attracted attention as a new therapeutic option for cancer. However, only a limited number of patients have responded to this treatment approach. In this study, we searched for compounds that enhance the efficacy of anti-PD-1 mAb using mixed lymphocyte reaction (MLR), which is a mixed culture system of the two key cells (dendritic and T cells) involved in tumor immunity. We found that amlexanox enhanced production of interferon (IFN)-γ, an indicator of T cell activation, by anti-PD-1 mAb. Amlexanox also induced PD-L1 expression in dendritic cells in MLR, whereas it did not stimulate interleukin-2 production by Jurkat T cells. These results suggest that amlexanox acts on dendritic cells, not T cells, in MLR. Furthermore, it enhanced the antitumor effect of the anti-PD-1 mAb in vivo in a mouse tumor-bearing model. The combination of amlexanox and anti-PD-1 mAb increased the expression of Ifng encoding IFN-γ, IFN-γ-related genes, Cd274 encoding PD-L1, and cytotoxic T cell-related genes in tumors. In conclusion, amlexanox stimulates the antitumor effect of anti-PD-1 mAb by acting on dendritic cells, which in turn activates cytotoxic T cells in tumors.

Myristate can be used as a carbon and energy source for the asymbiotic growth of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi, forming symbiotic associations with land plants, are obligate symbionts that cannot complete their natural life cycle without a host. The fatty acid auxotrophy of AM fungi is supported by recent studies showing that lipids synthesized by the host plants are transferred to the fungi, and that the latter lack genes encoding cytosolic fatty acid synthases. Therefore, to establish an asymbiotic cultivation system for AM fungi, we tried to identify the fatty acids that could promote biomass production. To determine whether AM fungi can grow on medium supplied with fatty acids or lipids under asymbiotic conditions, we tested eight saturated or unsaturated fatty acids (C12 to C18) and two ß-monoacylglycerols. Only myristate (C14:0) led to an increase in the biomass of Rhizophagus irregularis, inducing extensive hyphal growth and formation of infection-competent secondary spores. However, such spores were smaller than those generated symbiotically. Furthermore, we demonstrated that R. irregularis can take up fatty acids in its branched hyphae and use myristate as a carbon and energy source. Myristate also promoted the growth of Rhizophagus clarus and Gigaspora margarita Finally, mixtures of myristate and palmitate accelerated fungal growth and induced a substantial change in fatty acid composition of triacylglycerol compared with single myristate application, although palmitate was not used as a carbon source for cell wall biosynthesis in this culture system. Our findings demonstrate that myristate boosts the asymbiotic growth of AM fungi and can also serve as a carbon and energy source.

Activation of the VPAC2 Receptor Impairs Axon Outgrowth and Decreases Dendritic Arborization in Mouse Cortical Neurons by a PKA-Dependent Mechanism.

Clinical studies have shown that microduplications at 7q36.3, containing VIPR2, confer significant risk for schizophrenia and autism spectrum disorder (ASD). VIPR2 gene encodes the VPAC2 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Lymphocytes from patients with these mutations exhibited higher VIPR2 gene expression and VIP-induced cAMP responsiveness, but mechanisms by which overactive VPAC2 signaling may lead to these psychiatric disorders are unknown. We have previously found that repeated administration of a selective VPAC2 receptor agonist Ro25-1553 in the mouse during early postnatal development caused synaptic alterations in the prefrontal cortex and sensorimotor gating deficits. In this study, we aimed to clarify the effects of VPAC2 receptor activation on neurite outgrowth in cultured primary mouse cortical neurons. Ro25-1553 and VIP caused reductions in total numbers and lengths of both neuronal dendrites and axons, while PACAP38 facilitated elongation of dendrites, but not axons. These effects of Ro25-1553 and VIP were blocked by a VPAC2 receptor antagonist PG99-465 and abolished in VPAC2 receptor-deficient mice. Additionally, Ro25-1553-induced decreases in axon and dendritic outgrowth in wild-type mice were blocked by a protein kinase A (PKA) inhibitor H89, but not by a PKC inhibitor GF109203X or a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor U0126. PACAP38- induced facilitation of dendritic outgrowth was blocked by U0126. These results suggest that activation of the VPAC2 receptor impairs neurite outgrowth and decreases branching of cortical neurons by a PKA-dependent mechanism. These findings also imply that the VIPR2-linkage to mental health disorders may be due in part to deficits in neuronal maturation induced by VPAC2 receptor overactivation.

ERN1 and CYCLOPS coordinately activate NIN signaling to promote infection thread formation in Lotus japonicus.

Legumes engage in symbiosis with nitrogen-fixing soil bacteria, collectively called rhizobia, under nitrogen-limited conditions. In many legumes, the root invasion of rhizobia is mediated by infection threads (ITs), tubular invaginations of the host cell wall and plasma membrane, developed from infection foci of deformed root hairs. IT formation is regulated by a series of signal transduction in host root. Nodulation signals activate the host transcription factor (TF), CYCLOPS, which directly induces expression of two TF genes, ERF REQUIRED FOR NODULATION1 (ERN1) and NODULE INCEPTION (NIN), essential for IT development. Here, we explored the relationship among these three symbiotic TF genes in the model legume Lotus japonicus and examined how their interplay contributes to IT formation. qRT-PCR analysis showed that NIN expression induced by rhizobial infection was attenuated in ern1-1, and further declined in cyclops-3 ern1-1. ERN1 overexpression led to induction of NIN expression in cyclops-3 ern1-1 in the presence of rhizobia. Thus, in addition to CYCLOPS, ERN1 is able to increase the NIN expression level depending on infection. Furthermore, consistent with this transcriptional hierarchy, ectopic expression of ERN1 as well as NIN suppressed the IT-deficient cyclops-3 phenotype, but ERN1 failed to confer ITs in the nin-2 root. However, the ern1-1 symbiotic epidermal phenotype was not suppressed by the NIN ectopic expression. The cyclops-3 ern1-1 double mutant was less sensitive to rhizobial infection than the single mutants and defective in the symbiotic root hair response at earlier stages. This more severe phenotype of the double mutant suggests a role for ERN1 that independent of the CYCLOPS-mediated transcriptional regulation. We conclude that ERN1 is involved in regulating NIN expression in addition to CYCLOPS, and these TFs coordinately promote the symbiotic root hair response and IT development. Our data help to reveal the extensive role of ERN1 in root nodule symbiosis signaling.

Pharmacokinetic-Pharmacodynamic Modeling of Brain Dopamine Levels Based on Dopamine Transporter Occupancy after Administration of Methylphenidate in Rats.

Dopamine exerts various effects including movement coordination and reward. It is useful to understand the quantitative relationship between drug pharmacokinetics and target engagement such as the change in occupancy and dopamine level in brain for the proper treatment of dopamine-related diseases. This study was aimed at developing a pharmacokinetic-pharmacodynamic (PK-PD) model based on dopamine transporter (DAT) occupancies that could describe changes in extracellular dopamine levels in brain after administration of methylphenidate (a DAT inhibitor) to rat. First, uptake of fluorescent substrates was studied in DAT-expressing human embryonic kidney 293 cells and concentration dependently inhibited by methylphenidate. By analyzing the uptake of fluorescent substrates in the presence or absence of methylphenidate, a mathematical model could estimate the association and dissociation rate constants of methylphenidate for DAT. Next, we measured the concentrations of methylphenidate in plasma and cerebrospinal fluid (CSF) and extracellular dopamine levels in the nucleus accumbens after single intraperitoneal administration of methylphenidate. The concentrations of methylphenidate in plasma increased almost dose proportionally and the CSF-to-plasma concentration ratio was similar among evaluated dose. The extracellular dopamine levels also increased with dose. These data were analyzed using the mechanism-based PK-PD model, which incorporates dopamine biosynthesis, release from a synapse, reuptake via DAT into a synapse, and elimination from a synapse. Methylphenidate concentrations in plasma and dopamine profiles predicted by the PK-PD model were close to in vivo observations. In conclusion, our mechanism-based PK-PD model can accurately describe dopamine levels in the brain after administration of methylphenidate to rats.

In vivo hepatic clearance of lipophilic drugs predicted by in vitro uptake data into cryopreserved hepatocytes suspended in sera of rats, guinea pigs, monkeys and humans.

Evaluation of uptake of lipophilic acid compounds into hepatocytes was an unresolved drug development issue because of their adsorption to cells and materials and low analytical sensitivity and accuracy in assessment of protein bindings. Uptake assays of compounds using hepatocytes suspended in serum were expected to solve these problems for prediction of in vivo hepatic clearance. Here, for compounds with high protein binding (>99%), diflunisal, montelukast, cerivastatin, telmisartan, fluvastatin and six new drug candidates, in vivo hepatic clearance predicted based on hepatic depletion and uptake (CLh, uptake, predicted) data using hepatocytes in the absence and presence of sera was investigated. In vitro hepatic uptake results with hepatocytes suspended in serum improved prediction of human hepatic clearance values for highly lipophilic montelukast and telmisartan. In vivo CLh, uptake, predicted values of six new highly lipophilic acid drug candidates (protein binding >99.97%) and diflunisal, montelukast and cerivastatin predicted based on hepatocytes suspended in serum were within threefold differences of their total clearance in vivo in rats, guinea pigs or monkeys, except for montelukast in monkeys (5.8-fold). These results suggest that the human hepatic uptake in hepatocytes suspended in serum is useful for prediction of CLh, uptake, predicted, especially for highly lipophilic/protein binding acid compounds.

Pharmacokinetics and safety/efficacy of levodopa pro-drug ONO-2160/carbidopa for Parkinson's disease.

We conducted a phase I study investigating the efficacy, safety, and tolerability of ONO-2160, a newly developed levodopa pro-drug, and carbidopa compared with levodopa and carbidopa to stabilize levodopa plasma concentration fluctuations in Japanese patients with Parkinson's disease. In an open-label two-period design, patients (n = 12) with Parkinson's disease received levodopa and carbidopa for 3 days before 7 days of treatment with ONO-2160 and carbidopa. Patients were primarily evaluated using the Unified Parkinson's Disease Rating Scale Part III, a Parkinson's disease symptom diary, and analysis of adverse events. Pharmacokinetic analysis of plasma levodopa concentration was also performed. ONO-2160 and carbidopa therapy stabilized effective plasma levodopa concentration. No adverse events with safety concerns were observed. The combination of ONO-2160 and carbidopa produced a prolonged and stable plasma levodopa concentration with a reduction in Unified Parkinson's Disease Rating Scale Part III total scores. The combination was well tolerated, with no safety concerns, when administered to Japanese patients with Parkinson's disease.

Systemic administration of guanfacine improves food-motivated impulsive choice behavior primarily via direct stimulation of postsynaptic α2A-adrenergic receptors in rats.

Impulsive choice behavior, which can be assessed using the delay discounting task, is a characteristic of various psychiatric disorders, including attention-deficit/hyperactivity disorder (ADHD). Guanfacine is a selective α2A-adrenergic receptor agonist that is clinically effective in treating ADHD. However, there is no clear evidence that systemic guanfacine administration reduces impulsive choice behavior in the delay discounting task in rats. In the present study, we examined the effect of systemic guanfacine administration on food-motivated impulsive choice behavior in rats and the neuronal mechanism underlying this effect. Repeated administration of either guanfacine, methylphenidate, or atomoxetine significantly enhanced impulse control, increasing the number of times the rats chose a large but delayed reward in a dose-dependent manner. The effect of guanfacine was significantly blocked by pretreatment with an α2A-adrenergic receptor antagonist. Furthermore, the effect of guanfacine remained unaffected in rats pretreated with a selective noradrenergic neurotoxin, consistent with a post-synaptic action. In contrast, the effect of atomoxetine on impulsive choice behavior was attenuated by pretreatment with the noradrenergic neurotoxin. These results provide the first evidence that systemically administered guanfacine reduces impulsive choice behavior in rats and that direct stimulation of postsynaptic, rather than presynaptic, α2A-adrenergic receptors is involved in this effect.

Function and evolution of a Lotus japonicus AP2/ERF family transcription factor that is required for development of infection threads.

Legume-rhizobium symbiosis is achieved by two major events evolutionarily acquired: root hair infection and organogenesis. Infection thread (IT) development is a distinct element for rhizobial infection. Through ITs, rhizobia are efficiently transported from infection foci on root hairs to dividing meristematic cortical cells. To unveil this process, we performed genetic screening using Lotus japonicus MG-20 and isolated symbiotic mutant lines affecting nodulation, root hair morphology, and IT development. Map-based cloning identified an AP2/ERF transcription factor gene orthologous to Medicago truncatula ERN1. LjERN1 was activated in response to rhizobial infection and depended on CYCLOPS and NSP2. Legumes conserve an ERN1 homolog, ERN2, that functions redundantly with ERN1 in M. truncatula. Phylogenetic analysis showed that the lineages of ERN1 and ERN2 genes originated from a gene duplication event in the common ancestor of legume plants. However, genomic analysis suggested the lack of ERN2 gene in the L. japonicus genome, consistent with Ljern1 mutants exhibited a root hair phenotype that is observed in ern1/ern2 double mutants in M. truncatula. Molecular evolutionary analysis suggested that the nonsynonymous/synonymous rate ratios of legume ERN1 genes was almost identical to that of non-legume plants, whereas the ERN2 genes experienced a relaxed selective constraint.

Phosphate Treatment Strongly Inhibits New Arbuscule Development But Not the Maintenance of Arbuscule in Mycorrhizal Rice Roots.

Phosphorus (P) is a crucial nutrient for plant growth, but its availability to roots is limited in soil. Arbuscular mycorrhizal (AM) symbiosis is a promising strategy for improving plant P acquisition. However, P fertilizer reduces fungal colonization (P inhibition) and compromises mycorrhizal P uptake, warranting studies on the mechanistic basis of P inhibition. In this study, early morphological changes in P inhibition were identified in rice (Oryza sativa) using fungal cell wall staining and live-cell imaging of plant membranes that were associated with arbuscule life cycles. Arbuscule density decreased, and aberrant hyphal branching was observed in roots at 5 h after P treatment. Although new arbuscule development was severely inhibited, preformed arbuscules remained intact and longevity remained constant. P inhibition was accelerated in the rice pt11-1 mutant, which lacks P uptake from arbuscule branches, suggesting that mature arbuscules are stabilized by the symbiotic P transporter under high P condition. Moreover, P treatment led to increases in the number of vesicles, in which lipid droplets accumulated and then decreased within a few days. The development of new arbuscules resumed within by 2 d. Our data established that P strongly and temporarily inhibits new arbuscule development, but not intraradical accommodation of AM fungi.

A Successful Case of a Patient Undergoing Warfarin and S-1 Therapy Using Internet-based Control of Home-measured PT-INR.

  To avoid major bleeding events in warfarin and S-1 combination therapy, PT-INR levels should be monitored frequently to allow for precise adjustments of the warfarin dose and to verify any side effects reported by the patient. We therefore developed a support system where outpatients obtain a home-measured PT-INR value using the CoaguChek(®) system and submit it along with details of any side effects to us via the Internet using their mobile phone. A 59-year-old man was started on warfarin (1.5 mg/d) and S-1 (100 mg/d), a combination preparation of tegafur, gimeracil, and oteracil potassium, to treat cholangiocarcinoma. The patient sent his data to the hospital pharmacist every two days after starting S-1 therapy. When the PT-INR was outside the target range of 1.5-2.7, the pharmacist, after consulting the physician, instructed the patient to change his warfarin dose by 0.5 mg. On day 24 after starting S-1, PT-INR had increased from 1.6 to 2.8, so the dose was decreased by 0.5 mg. Thereafter, the dose was adjusted by 0.5-1.0 mg during the observation period so that the patient was able to maintain the therapeutic range approximately 90% of the time. We anticipate this system can be applied to S-1 which interact with warfarin, thereby enabling safer anticoagulation therapy.

Age-related changes of hepatic clearances of cytochrome P450 probes, midazolam and R-/S-warfarin in combination with caffeine, omeprazole and metoprolol in cynomolgus monkeys using in vitro-in vivo correlation.

1. Pharmacokinetics of human cytochrome P450 probes (caffeine, racemic warfarin, omeprazole, metoprolol and midazolam) were investigated after single intravenous and oral administrations at doses of 0.20 and 1.0 mg kg(-1), respectively, in combination to three young (3-year-old) and three aged (16-year-old) cynomolgus monkeys. 2. The plasma concentrations of caffeine and R-/S-warfarin decreased slowly in a monophasic manner, but those of omeprazole, metoprolol and midazolam decreased rapidly, in a similar manner to those as reported for pharmacokinetics in humans. 3. The mean maximum concentrations of R- and S-warfarin (4.6 and 3.7 µg/mL, respectively) in aged monkeys after oral administration were significantly higher than those in young monkeys (3.3 and 2.7 µg/mL). The mean clearance (CL) values of midazolam in aged monkeys (9.5 mL/min/kg) were significantly lower than those in young monkeys (13 mL/min/kg). 4. Individual intrinsic CL values for omeprazole (r = 0.29) and metoprolol (r = 0.30) of individual monkey livers were inversely correlated with their ages significantly (p < 0.05) in liver microsomes prepared from 55 cynomolgus monkeys. 5. These results suggest that cynomolgus monkeys could be a good model for humans, especially with particular characteristics in reduced CLs of some human P450 substrates by aging.

Sensitive and specific PCR systems for detection of both Chinese and Japanese severe fever with thrombocytopenia syndrome virus strains and prediction of patient survival based on viral load.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high case fatality risk and is caused by the SFTS virus (SFTSV). A retrospective study conducted after the first identification of an SFTS patient in Japan revealed that SFTS is endemic to the region, and the virus exists indigenously in Japan. Since the nucleotide sequence of Japanese SFTSV strains contains considerable differences compared with that of Chinese strains, there is an urgent need to establish a sensitive and specific method capable of detecting the Chinese and Japanese strains of SFTSV. A conventional one-step reverse transcription-PCR (RT-PCR) (cvPCR) method and a quantitative one-step RT-PCR (qPCR) method were developed to detect the SFTSV genome. Both cvPCR and qPCR detected a Chinese SFTSV strain. Forty-one of 108 Japanese patients suspected of having SFTS showed a positive reaction by cvPCR. The results from the samples of 108 Japanese patients determined by the qPCR method were in almost complete agreement with those determined by cvPCR. The analyses of the viral copy number level in the patient blood samples at the acute phase determined by qPCR in association with the patient outcome confirmed that the SFTSV RNA load in the blood of the nonsurviving patients was significantly higher than that of the surviving patients. Therefore, the cvPCR and qPCR methods developed in this study can provide a powerful means for diagnosing SFTS. In addition, the detection of the SFTSV genome level by qPCR in the blood of the patients at the acute phase may serve as an indicator to predict the outcome of SFTS.

Age-related pharmacokinetic changes of acetaminophen, antipyrine, diazepam, diphenhydramine, and ofloxacin in male cynomolgus monkeys and beagle dogs.

1. The pharmacokinetics of acetaminophen (marker of gastric emptying), antipyrine (marker of hepatic metabolic activity and total body water), diazepam (lipophilic and highly distributed), diphenhydramine (hepatic blood flow-limited and alpha-1 acid glycoprotein bound) and ofloxacin (renally eliminated) were evaluated in cynomolgus monkeys (3-18 years old) and beagle dogs (2-11 years old) as models in elderly persons. 2. Gastric pH fluctuated with aging in monkeys and dogs. The concentration of alpha-1 acid glycoprotein appeared to be increased by aging. There were no age-related differences in the absorption rates of the drugs under the conditions used in the study. Total body fat increased and water decreased in monkeys, but these parameters did not change in dogs. 3. Hepatic blood flow decreased in both species, but a significant decrease of hepatic clearance was only seen in monkeys. Renal clearance decreased significantly with age in monkeys and showed a tendency to decrease in dogs. 4. Age-related alterations of physiological parameters in monkeys are in agreement with clinical observations in humans, except for the lack of a change in the plasma albumin concentration. Therefore, this study suggests that monkey might be a suitable animal model for prediction of age-related changes in pharmacokinetics in humans.

Assessment of absorption potential of poorly water-soluble drugs by using the dissolution/permeation system.

This study aims to assess the absorption potential of oral absorption of poorly water-soluble drugs by using the dissolution/permeation system (D/P system). The D/P system can be used to perform analysis of drug permeation under dissolution process and can predict the fraction of absorbed dose in humans. When celecoxib at 1/100 of a clinical dose was applied to the D/P system, percentage of dose dissolved and permeated significantly decreased with an increase in the applied amount, resulting in the oral absorption being predicted to be 22-55%. Whereas similar dissolution and permeation profiles of montelukast sodium were observed, estimated absorption (69-85%) was slightly affected. Zafirlukast absorption (33-36%) was not significantly affected by the dose, although zafirlukast did not show complete dissolution. The relationship between clinical dose and predicted oral absorption of drugs corresponded well to clinical observations. The limiting step of the oral absorption of celecoxib and montelukast sodium was solubility, while that of zafirlukast was dissolution rate. However, due to high permeability of montelukast, oral absorption was not affected by dose. Therefore, the D/P system is a useful tool to assess the absorption potential of poorly water-soluble drugs for oral use.

Too much love, a novel Kelch repeat-containing F-box protein, functions in the long-distance regulation of the legume-Rhizobium symbiosis.

The interaction of legumes with N2-fixing bacteria collectively called rhizobia results in root nodule development. The number of nodules formed is tightly restricted through the systemic negative feedback control by the host called autoregulation of nodulation (AON). Here, we report the characterization and gene identification of TOO MUCH LOVE (TML), a root factor that acts during AON in a model legume Lotus japonicus. In our genetic analyses using another root-regulated hypernodulation mutant, plenty, the tml-1 plenty double mutant showed additive effects on the nodule number, whereas the tml-1 har1-7 double mutant did not, suggesting that TML and PLENTY act in different genetic pathways and that TML and HAR1 act in the same genetic pathway. The systemic suppression of nodule formation by CLE-RS1/RS2 overexpression was not observed in the tml mutant background, indicating that TML acts downstream of CLE-RS1/RS2. The tml-1 Snf2 double mutant developed an excessive number of spontaneous nodules, indicating that TML inhibits nodule organogenesis. Together with the determination of the deleted regions in tml-1/-2/-3, the fine mapping of tml-4 and the next-generation sequencing analysis, we identified a nonsense mutation in the Kelch repeat-containing F-box protein. As the gene knockdown of the candidate drastically increased the number of nodules, we concluded that it should be the causative gene. An expression analysis revealed that TML is a root-specific gene. In addition, the activity of ProTML-GUS was constitutively detected in the root tip and in the nodules/nodule primordia upon rhizobial infection. In conclusion, TML is a root factor acting at the final stage of AON.