Type 2C protein phosphatase clade D family members dephosphorylate guard cell plasma membrane H+-ATPase.

Plasma membrane (PM) H+-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting stomatal opening. Previous in vitro biochemical investigation suggested that Mg2+- and Mn2+-dependent membrane-localized type 2C protein phosphatase (PP2C)-like activity mediates the dephosphorylation of PM H+-ATPase in guard cells. PP2C clade D (PP2C.D) was later demonstrated to be involved in PM H+-ATPase dephosphorylation during auxin-induced cell expansion in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether PP2C.D phosphatases are involved in PM H+-ATPase dephosphorylation in guard cells. Transient expression experiments using Arabidopsis mesophyll cell protoplasts revealed that all PP2C.D isoforms dephosphorylate the endogenous PM H+-ATPase. We further analyzed PP2C.D6/8/9, which display higher expression levels than other isoforms in guard cells, observing that pp2c.d6, pp2c.d8, and pp2c.d9 single mutants showed similar light-induced stomatal opening and phosphorylation status of PM H+-ATPase in guard cells as Col-0. In contrast, the pp2c.d6/9 double mutant displayed wider stomatal apertures and greater PM H+-ATPase phosphorylation in response to blue light, but delayed dephosphorylation of PM H+-ATPase in guard cells; the pp2c.d6/8/9 triple mutant showed similar phenotypes to those of the pp2c.d6/9 double mutant. Taken together, these results indicate that PP2C.D6 and PP2C.D9 redundantly mediate PM H+-ATPase dephosphorylation in guard cells. Curiously, unlike auxin-induced cell expansion in seedlings, auxin had no effect on the phosphorylation status of PM H+-ATPase in guard cells.

Involvement of α-galactosidase OmAGAL2 in planteose hydrolysis during seed germination of Orobanche minor.

Root parasitic weeds of the Orobanchaceae, such as witchweeds (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.), cause serious losses in agriculture worldwide, and efforts have been made to control these parasitic weeds. Understanding the characteristic physiological processes in the life cycle of root parasitic weeds is particularly important to identify specific targets for growth modulators. In our previous study, planteose metabolism was revealed to be activated soon after the perception of strigolactones in germinating seeds of O. minor. Nojirimycin inhibited planteose metabolism and impeded seed germination of O. minor, indicating a possible target for root parasitic weed control. In the present study, we investigated the distribution of planteose in dry seeds of O. minor by matrix-assisted laser desorption/ionization-mass spectrometry imaging. Planteose was detected in tissues surrounding-but not within-the embryo, supporting its suggested role as a storage carbohydrate. Biochemical assays and molecular characterization of an α-galactosidase family member, OmAGAL2, indicated that the enzyme is involved in planteose hydrolysis in the apoplast around the embryo after the perception of strigolactones, to provide the embryo with essential hexoses for germination. These results indicate that OmAGAL2 is a potential molecular target for root parasitic weed control.

Augmented leptin-induced trefoil factor 3 expression and epidermal growth factor receptor transactivation differentially influences neoplasia progression in the stomach and colorectum of dietary fat-induced obese mice.

Obesity is a risk factor for gastrointestinal malignancies and tumors. However, which factors either protect or predispose the gastrointestinal organs to high-fat diet (HFD)-induced neoplasia remains unclear. Here, we demonstrate that HFD impacts the stomach to a greater extent as compared to the colorectum, resulting in leptin receptor (LepR) signaling-mediated neoplasia in the tissues. HFD activated leptin signaling, which in turn, accelerates the pathogenesis in the gastric mucosa more than that in the colorectum along with ectopic TFF3 expression. Moreover, in the stomach, higher levels of phosphorylated epidermal growth factor receptor (EGFR) in addition to the activation of STAT3 and Akt were observed as compared to the colorectum. The mice with LepR deletion in the gastrointestinal epithelium exhibited a suppressed induction of leptin, TFF3, and phosphorylated EGFR in the stomach, whereas the levels in the colorectum were insignificant. In co-transfected COS-7 cells with LepR and EGFR plasmid DNA, leptin transactivated EGFR to accelerate TFF3 induction along with activation of STAT3, ERK1/2, Akt, and PI3K p85/p55. Furthermore, TFF3 could bind to EGFR but did not transactivate LepR. Leptin-induced TFF3 induction was markedly suppressed by inhibitors of PI3K (LY294002) and EGFR (Erlotinib). Together, these results suggest a novel role of LepR-mediated signaling in transactivating EGFR that leads to TFF3 expression via the PI3K-Akt pathway. Therefore, this study sheds light on the identification of potentially new therapeutic targets for the treatment of pre-cancerous symptoms in stomach and colorectum.

Challenging Approach to the Development of Novel Estrogen Receptor Modulators Based on the Chemical Properties of Guaiazulene.

Tamoxifen, a therapeutic agent for breast cancer, has been associated with genetic polymorphisms in the metabolism of N,N-dialkylaminoethyl substituent, which plays an important role in the expression of selective estrogen receptor modulator (SERM) activity. To solve this problem, we developed a novel estrogen receptor (ER) modulator, Az-01, on the basis of the aromaticity, dipole moment, and isopropyl group of guaiazulene. Az-01 showed four-fold lower binding affinity for ER than E2 but had similar ER-binding affinity to that of 4-hydroxytamoxifen (4-HOtam). Unlike tamoxifen, Az-01 acted as a partial agonist with very weak estrogenic activity at high concentrations when used alone, and it showed potent anti-estrogenic activity in the presence of E2. The cell proliferation and inhibition activities of Az-01 were specific to ER-expressing MCF-7 cells, and no effect of Az-01 on other cell proliferation signals was observed. These findings are important for the development of new types of SERMs without the N,N-dialkylaminoethyl substituent as a privileged functional group for SERMs.

Constitutive BiP protein accumulation in Arabidopsis mutants defective in a gene encoding chloroplast-resident stearoyl-acyl carrier protein desaturase.

The unfolded protein response (UPR) occurs when protein folding and maturation are disturbed in the endoplasmic reticulum (ER). During the UPR, a number of genes including those encoding ER-resident molecular chaperones are induced. In Arabidopsis, BiP3 has been used as a UPR marker gene whose expression is strongly induced in response to ER stress. In this study, we mutagenized Arabidopsis plants expressing ß-glucuronidase (GUS) gene under the control of BiP3 promoter and isolated a mutant that exhibits strong GUS activity without treatment with ER stress inducers. By whole genome sequencing, we identified a causal gene in the mutant as SUPPRESSOR OF SALICYLIC ACID INSENSITIVITY2 (SSI2), which encodes stearoyl-acyl carrier protein desaturase that converts stearic acids to oleic acids in the chloroplasts. In addition to GUS proteins, the ssi2 mutant accumulates endogenous BiP3 proteins without treatment by any stress reagents. Interestingly, although the degree of endogenous BiP3 protein accumulation in the ssi2 mutant was comparable to that in wild-type plants treated with the ER stress inducer tunicamycin, much less BiP3 transcripts were detected in the ssi2 mutant compared to tunicamycin-treated wild-type plants. Our finding suggests a genetic link between fatty acid metabolism in the chloroplasts and ER functions.

ER subtype selectivity of m-carborane-containing phenols: C-alkyl groups on the m-carborane cage enhance ERα selectivity.

Estrogen receptor (ER) exhibits two subtypes, ERα and ERß, whose biological functions are quite different despite expression in the same tissues. We developed diiodo-m-carborane derivative 3a, which showed 14-fold selectivity for ERß with high binding affinity toward ERß. Interestingly, introduction of an alkyl group into the carbon atom of the m-carborane cage of 3a markedly enhanced the binding affinity toward ERα and decreased affinity toward ERß. C-n-propyl derivative 3d showed 28-fold selectivity for ERα in an ER binding assay and promoted proliferation of MCF-7 breast cancer cells. Docking simulation studies suggest that the directions of the n-propyl group and the diiodo substituent introduced on the m-carborane cage play important roles for the control of ER subtype selectivity. As 3a and 3d showed ERß and ERα selectivity with high binding affinity, respectively, these ligands may be useful as biological tools to aid in understanding the different roles of ER subtypes.

Design and Synthesis of Novel Breast Cancer Therapeutic Drug Candidates Based upon the Hydrophobic Feedback Approach of Antiestrogens.

Based upon hydrophobic feedback approaches, we designed and synthesized novel sulfur-containing ERα modulators (4 and 5) as breast cancer therapeutic drug candidates. The tetrahydrothiepine derivative 5a showed the highest binding affinity toward ERα because of its high hydrophobicity, and it acted as an agonist toward MCF-7 cell proliferation. The corresponding alkylamino derivative 5d maintained high binding affinity to ERα and potently inhibited MCF-7 cell proliferation (IC50: 0.09 µM). Docking simulation studies of compound 5d with the ERα BD revealed that the large hydrophobic moiety of compound 5d fit well into the hydrophobic pocket of the ERα LBD and that the sulfur atom of compound 5d formed a sulfur-π interaction with the amino acid residue His524 of the ERα LBD. These interactions play important roles for the binding affinity of compound 5d to the ERα LBD.

Consistency of supplied food and dentition status of the elderly in residential care homes.

BACKGROUND: The association between oral health and malnutrition has been investigated in detail. The nutrition of elderly subjects in residential care homes is determined by caregivers, dietitians or nutritionists and managed by changing the consistency of their supplied food. However, few reports have described the relationship between oral condition and supplied food consistency. The objective of this study was to determine dentition status and care levels that correlate with supplied food consistency among elderly residents of care facilities. In addition, we estimated the care level at which ordinary food consistency can be supplied by caregivers who cannot diagnose dental status. METHOD: Several factors, including dentition, wearing removable dentures, meals categorized as ordinary or processed (sliced, mashed, or liquefied), and care levels according to the Japanese standardized care-needs certification system were investigated in 276 elderly residents (male, n = 56; female, n = 220; mean age, 87.68 ± 5.94 years) of 12 fee-based care facilities. RESULTS: The results of this study showed that care levels were significantly correlated with the consistency of the food supplied to the residents. When supplied food consistency was categorized as ordinary or processed, the number of remaining teeth and the number of tooth contact pairs, either natural or artificial, were statistically significant. From logistic regression analysis, it was determined that the numbers of tooth contact pairs were statistically significant among residents requiring high levels of care. CONCLUSION: The number of tooth contact pairs, either natural or artificial, was one of the contributing factors for deciding supplied food consistency among elderly residents of care facilities. Elderly residents requiring less than care level 3 should have ordinary meals.

Pre-endodontic Post and Core Technique for Endodontic and Prosthodontic Treatment.

INTRODUCTION: Displacement of provisional fixed prostheses may result in undesirable and embarrassing outcomes in dental treatments, especially in endodontic treatment. Development of certain counter measures has been necessary to avoid such discomforts. AIM: The aim of this report was to propose a pre-endodontic post and core technique to achieve smooth progress of the treatment. MATERIALS AND METHODS: The patient was a 59-year-old male diagnosed with an infraocclusion caused by wear of his teeth. He received full mouth provisional fixed restorations for a complete oral rehabilitation. Displacement and fracture of the restorations frequently occurred during the observation period for the function of the restorations. Therefore, the pre-endodontic post and core technique was applied to the abutment teeth before their endodontic treatments were started. The technique consisted of three steps as follows: Step 1: Caries removal and dowel preparation were performed for the abutment teeth having apical periodontitis. Composite cores were indirectly fabricated, which had access holes for endodontic treatment. Step 2: The cores were bonded to the teeth. In endodontic treatment, rubber dam appliances were easily placed owing to the core, and proper tooth isolation was accomplished. Step 3: Fiberposts were bonded to the dowel holes through the access holes after the root canal filling. During endodontic treatment, displacement and/or fracture of the provisional restorations did not occur. CONCLUSION: The pre-endodontic post and core technique was effective in obtaining improved retention of provisional restoration, appropriate isolation for endodontic treatment, and sufficient retention of the post and core. CLINICAL SIGNIFICANCE: The pre-endodontic post and core technique is useful for avoiding the discomforts in dental treatments, namely, a smooth transition from endodontic to prosthodontic treatment can be achieved.

Design and synthesis of iodocarborane-containing ligands with high affinity and selectivity toward ERß.

The selectivity and the binding affinity of previously reported carborane-containing ligands 2 and 3 toward ERß remains to be optimized. To improve their biological profiles, a series of iodinated carboranyl phenol derivatives (4-6) were designed and synthesized as prospective ERß-selective ligands with high affinity. Several iodinated carboranyl phenols showed high relative binding affinity (RBA) values for both ERs, and especially for ERß, due to suitable hydrophobic interactions of the iodine atoms with the hydrophobic amino acid residues of the ERß ligand-binding domains. Among these derivatives, 9,10-diiodo-m-carborane 5f exhibited a more than 100% increase of the RBA values toward ERß, a 14-fold increased selectivity for ERß over ERα, and ER-agonistic activity in MCF-7 cell proliferation assays.

The effect of rapamycin on biodiesel-producing protist Euglena gracilis.

Rapamycin induces autophagy with lipid remodeling in yeast and mammalian cells. To investigate the lipid biosynthesis of Euglena gracilis, rapamycin was supplemented in comparison with two model algae, Chlamydomonas reinhardtii and Cyanidioschyzon merolae. In Euglena, rapamycin induced the reduction of chlorophylls and the accumulation of neutral lipids without deterring its cell proliferation. Its lipidomic profile revealed that the fatty acid composition did not alter by supplementing rapamycin. In Chlamydomonas, however, rapamycin induced serious growth inhibition as reported elsewhere. With a lower concentration of rapamycin, the alga accumulated neutral lipids without reducing chlorophylls. In Cyanidioschyzon, rapamycin did not increase neutral lipids but reduced its chlorophyll content. We also tested fatty acid elongase inhibitors such as pyroxasulfone or flufenacet in Euglena with no significant change in its neutral lipid contents. In summary, controlled supplementation of rapamycin can increase the yield of neutral lipids while the scheme is not always applicable for other algal species.

Clinical Usefulness of Mandibular Reconstruction Using Custom-Made Titanium Mesh Tray and Autogenous Particulate Cancellous Bone and Marrow Harvested From Tibia and/or Ilia.

The objective of this study was to evaluate usefulness of mandibular reconstructions using custom-made titanium mesh (Ti-mesh) tray and particulate cancellous bone and marrow (PCBM). Consecutive 21 patients who underwent mandibular reconstruction were enrolled in this study. They were 13 men and 8 women (mean age, 52.0 years). Virtual reality simulation was performed using computer software based on the preoperative computed tomography data. A 3-dimensional skull model was constructed using 3-dimensional printer. A tray was custom-made from Ti-mesh sheet bent to adapt to the model. After PCBM harvesting from posterior ilia and/or proximal tibia, the tray was fixed to the host bone. New bone formation and configuration of the reconstructed mandible were assessed radiologically. Complications were recorded in each patient during the follow-up period. Patients' satisfaction with postoperative facial contour was evaluated using visual analog scale (VAS score, range, 0-100). In 16 of 21 patients, excellent new bone formation was recognized and expected results were radiologically achieved. In 5 patients, new bone formation was insufficient. Causes of insufficient bone formation included postoperative infection in 2 patients, Ti-mesh tray fracture in 2 patients, and local recurrence of lower gingival cancer in 1 patient. To prevent a tray fracture, a double-layered Ti-mesh tray was useful. Mean VAS score on patients' satisfaction was 77.1. Our results comprehensively suggest that mandibular reconstruction using custom-made Ti-mesh tray and PCBM is clinically useful.

Novel plant immune-priming compounds identified via high-throughput chemical screening target salicylic acid glucosyltransferases in Arabidopsis.

Plant activators are compounds, such as analogs of the defense hormone salicylic acid (SA), that protect plants from pathogens by activating the plant immune system. Although some plant activators have been widely used in agriculture, the molecular mechanisms of immune induction are largely unknown. Using a newly established high-throughput screening procedure that screens for compounds that specifically potentiate pathogen-activated cell death in Arabidopsis thaliana cultured suspension cells, we identified five compounds that prime the immune response. These compounds enhanced disease resistance against pathogenic Pseudomonas bacteria in Arabidopsis plants. Pretreatments increased the accumulation of endogenous SA, but reduced its metabolite, SA-O-ß-d-glucoside. Inducing compounds inhibited two SA glucosyltransferases (SAGTs) in vitro. Double knockout plants that lack both SAGTs consistently exhibited enhanced disease resistance. Our results demonstrate that manipulation of the active free SA pool via SA-inactivating enzymes can be a useful strategy for fortifying plant disease resistance and may identify useful crop protectants.

Time Course of Behavioral Alteration and mRNA Levels of Neurotrophic Factor Following Stress Exposure in Mouse.

Stress is known to affect neurotrophic factor expression, which induces depression-like behavior. However, whether there are time-dependent changes in neurotrophic factor mRNA expression following stress remains unclear. In the present study, we tested whether chronic stress exposure induces long-term changes in depression-related behavior, serum corticosterone, and hippocampal proliferation as well as neurotrophic factor family mRNA levels, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and ciliary neurotrophic factor (CNTF), in the mouse hippocampus. The mRNA level of neurotrophic factors (BDNF, NGF, NT-3, and CNTF) was measured using the real-time PCR. The serum corticosterone level was evaluated by enzyme-linked immunosorbent assay, and, for each subject, the hippocampal proliferation was examined by 5-bromo-2-deoxyuridine immunostaining. Mice exhibited depression-like behavior in the forced-swim test (FST) and decreased BDNF mRNA and hippocampal proliferation in the middle of the stress exposure. After 15 days of stress exposure, we observed increased immobility in the FST, serum corticosterone levels, and BDNF mRNA levels and degenerated hippocampal proliferation, maintained for at least 2 weeks. Anhedonia-like behavior in the sucrose preference test and NGF mRNA levels were decreased following 15 days of stress. NGF mRNA levels were significantly higher 1 week after stress exposure. The current data demonstrate that chronic stress exposure induces prolonged BDNF and NGF mRNA changes and increases corticosterone levels and depression-like behavior in the FST, but does not alter other neurotrophic factors or performance in the sucrose preference test.

Synthesis and biological evaluation of novel m-carborane-containing estrogen receptor partial agonists as SERM candidates.

We designed and synthesized novel m-carborane-containing selective estrogen receptor modulator (SERM) candidates using previously reported m-carborane-containing ER partial agonist 1 as the lead compound. Biological activities were evaluated by means of ERα competitive binding assay and MCF-7 cell proliferation assay. Re-positioning the N,N-dimethylaminoethyloxy group at the para position of 1 to the meta position enhanced the ERα-binding affinity, and 4c showed the highest relative binding affinity (RBA: 83 vs 17ß-estradiol = 100) among the tested compounds. Compound 4b showed the most potent ER-agonist activity (EC50: 1.4 nM) and the lowest maximal efficacy (Emax: 50%) in MCF-7 cell proliferation assay. Inhibition of 0.1 nM 17ß-estradiol-induced MCF-7 cell proliferation by 4b (IC50: 0.4 µM) was at least 10 times more potent than that of the lead compound 1.

Design and synthesis of carborane-containing estrogen receptor-beta (ERß)-selective ligands.

Candidates for highly selective estrogen receptor-beta (ERß) ligands (6a-c, 7a-c, 8a and 8b) were designed and synthesized based on carborane-containing ER ligands 1 and 2 as lead compounds. Among them, p-carboranylcyclohexanol derivatives 8a and 8b exhibited high ERß selectivity in competitive binding assay: for example, 8a showed 56-fold selectivity for ERß over ERα. Docking studies of 8a and 8b with the ERα and ERß ligand-binding domains (LBDs) suggested that the p-carborane cage of the ligands is located close to key amino acid residues that influence ER-subtype selectivity, that is, Leu384 in the ERα LBD and Met336 in the ERß LBD. The p-carborane cage in 8a and 8b appears to play a crucial role in the increased ERß selectivity.

Streptococcus panodentis sp. nov. from the oral cavities of chimpanzees.

Three strains TKU9, TKU49 and TKU50(T) , were isolated from the oral cavities of chimpanzees (Pan troglodytes). The isolates were all gram-positive, facultative anaerobic cocci that lacked catalase activity. Analysis of partial 16S rRNA gene sequences showed that the most closely related species was Streptococcus infantis (96.7%). The next most closely related species to the isolates were S. rubneri, S. mitis, S. peroris and S. australis (96.6 to 96.4%). Based on the rpoB and gyrB gene sequences, TKU50(T) was clustered with other member of the mitis group. Enzyme activity and sugar fermentation patterns differentiated this novel bacterium from other members of the mitis group streptococci. The DNA G + C content of strain TKU50(T) was 46.7 mol%, which is the highest reported value for members of the mitis group (40-46 mol%). On the basis of the phenotypic characterization, partial 16S rRNA gene and sequences data for two housekeeping gene (gyrB and rpoB), we propose a novel taxa, S. panodentis for TKU 50(T) (type strain = CM 30579(T) = DSM 29921(T) ), for these newly described isolates.

Estrogenic activity of bis(4-hydroxyphenyl)methanes with cyclic hydrophobic structure.

Monoalkylated bis(4-hydroxyphenyl)methanes (e.g., 1) are reported to show weak binding affinity for estrogen receptor (ER). We hypothesized that introduction of appropriately located hydrophobic substituents in these compounds would increase the binding affinity. Indeed, we found that bis(4-hydroxyphenyl)methane bearing a 3,3-dimethylcyclohexyl group (7) shows potent ERα binding affinity, comparable to that of estradiol. Bulkier substituents could be introduced at the 3,3-position without decreasing the affinity. However, the position of the substituents was critical: the 4,4-dimethylcyclohexyl derivative (2) showed very weak binding affinity. The compounds with high ER-binding affinity showed predominantly agonistic activity, together with weak antagonistic activity at high concentration, in cell proliferation assay with human breast cancer cell line MCF-7. Further structure-function studies of these compounds and their derivatives might lead to the development of more selective and potent estrogen receptor modulators.

Surgical Intervention for Masticatory Muscle Tendon-Aponeurosis Hyperplasia Based on the Diagnosis Using the Four-Dimensional Muscle Model.

OBJECTIVES: The surgical target of Masticatory muscle tendon-aponeurosis hyperplasia (MMTAH) is the masseter or temporal muscle. In our clinic, the 4-dimentional muscle model (4DMM) has been used to decide if we should approach to the masseter or temporal muscle. The aim of this study is validate the clinical usefulness of 4DMM on the basis of the surgical results. METHODS: The 4DMM was constructed from the digital data of 3D-CT and 4-dimentional mandibular movements of the patients. It made us to able to visually observe the expansion rate of masticatory muscles at maximum mouth opening comparing to their length at closed mouth position. Fifteen patients were applied the 4DMM before the surgical treatment and 2 healthy volunteers were enrolled as control group. RESULTS: The expansion rate of temporal muscle at the maximum mouth opening in the patient group was significantly less than that in the control group (P < 0.05). On the other hand, the masseter muscles of all patients were expanded as same as the control group. Therefore the main cause of limitation of mouth-opening was suggested to be a contracture of the temporal muscle. Consequently, we performed successful bilateral coronoidectomy with no surgical intervention to the masseter muscles in all patients. CONCLUSION: The present 4DMM would be valuable modality to decide the target muscle of surgical treatment for patients with MMTAH. In this pathology, contracture of the temporal muscle seems to be main cause of limited mouth opening.

Novel estrogen receptor (ER) modulators containing various hydrophobic bent-core structures.

We previously discovered m-carborane-containing estrogen receptor (ER) modulator 4, which exhibits weak ER-agonistic and antagonistic activities in transactivation assays. With the aim of developing novel ER partial agonists, we designed and synthesized various analogues of 4 with a bent-core structure, that is, pseudo cyclic structure (5), tetrahydropyrimidinone (6), m-benzene (7), adamantane (8), and 9,10-dimethyl-m-carborane (9), in place of the m-carborane moiety. Compound 9 showed greater binding affinity than 4 in ER-binding assay using [6,7-(3)H]-17ß-estradiol and was a more effective partial agonist than 4 in MCF-7 cell proliferation assay. It appears to be a promising candidate as a selective ER modulator (SERM).