Enantioselective One-Pot Synthesis of Cyclopropane-Fused Tetrahydroquinolines via a Ru-Catalyzed Intramolecular Cyclopropanation.

A highly enantioselective one-pot synthesis of cyclopropane-fused tetrahydroquinolines bearing carbonyl functionalities, which are difficult to synthesize using conventional methods, is reported. Employing readily accessible alkene-tethered anthranilaldehydes, hydrazone formation and subsequent Ru-catalyzed intramolecular cyclopropanation furnish the desired products in ≤87% yield and ≤95% ee under mild conditions. Various anthranilaldehydes, functionalized alkenes, and N-aryl sulfonyl groups are tolerated, and a series of synthetic transformations were conducted to demonstrate the practical utility.

Coregulation of glutamine synthetase1;2 (GLN1;2) and NADH-dependent glutamate synthase (GLT1) gene expression in Arabidopsis roots in response to ammonium supply.

Ammonium absorbed by roots is assimilated into amino acids. The glutamine synthetase/glutamate synthase (glutamine 2-oxoglutarate aminotransferase) (GS/GOGAT) cycle is essential to this biological process. In Arabidopsis thaliana, GLN1;2 and GLT1 are the GS and GOGAT isoenzymes induced in response to ammonium supply and playing key roles in ammonium utilization. Although recent studies suggest gene regulatory networks involved in transcriptional regulation of ammonium-responsive genes, direct regulatory mechanisms for ammonium-induced expression of GS/GOGAT remain unclear. In this study, we revealed that the expression of GLN1;2 and GLT1 in Arabidopsis is not directly induced by ammonium but is regulated by glutamine or post-glutamine metabolites produced by ammonium assimilation. Previously, we identified a promoter region required for ammonium-responsive expression of GLN1;2. In this study, we further dissected the ammonium-responsive region of the GLN1;2 promoter and also performed a deletion analysis of the GLT1 promoter, which led to the identification of a conserved ammonium-responsive region. Yeast one-hybrid screening using the ammonium-responsive region of the GLN1;2 promoter as a decoy sequence revealed a trihelix family transcription factor DF1 that binds to this region. A putative DF1 binding site was also found in the ammonium-responsive region of the GLT1 promoter.

Specific Mutations near the Amyloid Precursor Protein Cleavage Site Increase γ-Secretase Sensitivity and Modulate Amyloid-ß Production.

Amyloid-ß peptides (Aßs) are produced via cleavage of the transmembrane region of the amyloid precursor protein (APP) by γ-secretase and are responsible for Alzheimer's disease. Familial Alzheimer's disease (FAD) is associated with APP mutations that disrupt the cleavage reaction and increase the production of neurotoxic Aßs, i.e., Aß42 and Aß43. Study of the mutations that activate and restore the cleavage of FAD mutants is necessary to understand the mechanism of Aß production. In this study, using a yeast reconstruction system, we revealed that one of the APP FAD mutations, T714I, severely reduced the cleavage, and identified secondary APP mutations that restored the cleavage of APP T714I. Some mutants were able to modulate Aß production by changing the proportions of Aß species when introduced into mammalian cells. Secondary mutations include proline and aspartate residues; proline mutations are thought to act through helical structural destabilization, while aspartate mutations are thought to promote interactions in the substrate binding pocket. Our results elucidate the APP cleavage mechanism and could facilitate drug discovery.

Supplemental Clostridium butyricum MIYAIRI 588 Affects Intestinal Bacterial Composition of Finishing Pigs.

Animal gastrointestinal tracts are populated by highly diverse and complex microbiotas. The gut microbiota influences the bioavailability of dietary components and is closely associated with physiological processes in the host. Clostridium butyricum reportedly improves growth performance and affects the gut microbiota and immune functions in post-weaning piglets. However, the effects of C. butyricum on finishing pigs remain unclear. Therefore, we herein investigated the effects of C. butyricum MIYAIRI 588 (CBM588) on the gut microbiota of finishing pigs. 16S rRNA gene sequencing was performed using fecal samples and ileal, cecal, and colonic contents collected after slaughtering. The α-diversity of the small intestinal microbiota was lower than that of the large intestinal microbiota, whereas ß-diversity showed different patterns depending on sample collection sites. The administration of CBM588 did not significantly affect the α- or ß-diversity of the microbiotas of fecal and intestinal content samples regardless of the collection site. However, a linear discriminant ana-lysis Effect Size revealed that the relative abundance of Lactobacillaceae at the family level, Bifidobacterium at the order level, and Lactobacillus ruminis and Bifidobacterium pseudolongum at the species level were higher in the fecal samples and cecal and colonic contents of the treatment group than in those of the control group. Therefore, the administration of CBM588 to finishing pigs affected the composition of the gut microbiota and increased the abundance of bacteria that are beneficial to the host. These results provide important insights into the effects of probiotic administration on relatively stable gut microbial ecosystems.

Specific Mutations in Aph1 Cause γ-Secretase Activation.

Amyloid beta peptides (Aßs) are generated from amyloid precursor protein (APP) through multiple cleavage steps mediated by γ-secretase, including endoproteolysis and carboxypeptidase-like trimming. The generation of neurotoxic Aß42/43 species is enhanced by familial Alzheimer's disease (FAD) mutations within the catalytic subunit of γ-secretase, presenilin 1 (PS1). FAD mutations of PS1 cause partial loss-of-function and decrease the cleavage activity. Activating mutations, which have the opposite effect of FAD mutations, are important for studying Aß production. Aph1 is a regulatory subunit of γ-secretase; it is presumed to function as a scaffold of the complex. In this study, we identified Aph1 mutations that are active in the absence of nicastrin (NCT) using a yeast γ-secretase assay. We analyzed these Aph1 mutations in the presence of NCT; we found that the L30F/T164A mutation is activating. When introduced in mouse embryonic fibroblasts, the mutation enhanced cleavage. The Aph1 mutants produced more short and long Aßs than did the wild-type Aph1, without an apparent modulatory function. The mutants did not change the amount of γ-secretase complex, suggesting that L30F/T164A enhances catalytic activity. Our results provide insights into the regulatory function of Aph1 in γ-secretase activity.

Specific mutations in presenilin 1 cause conformational changes in γ-secretase to modulate amyloid ß trimming.

γ-Secretase generates amyloid beta peptides (Aß) from amyloid precursor protein through multistep cleavages, such as endoproteolysis (ε-cleavage) and trimming (γ-cleavage). Familial Alzheimer's disease (FAD) mutations within the catalytic subunit protein of presenilin 1 (PS1) decrease γ-cleavage, resulting in the generation of toxic, long Aßs. Reducing long Aß levels has been proposed as an AD therapeutic strategy. Previously, we identified PS1 mutations that are active in the absence of nicastrin (NCT) using a yeast γ-secretase assay. Here, we analysed these PS1 mutations in the presence of NCT, and found that they were constitutively active in yeast. One triple, 13 double, and 5 single mutants enhanced ε-cleavage activity up to 2.7-fold. Furthermore, L241I, F411Y, S438P and F441L mutations modulated trimming activities to produce more short-Aß in yeast microsomes. When introduced in mouse embryonic fibroblasts, these mutations possessed similar or reduced ε-cleavage activity. However, two mutations, L241I and S438P, modulated trimming activities and changed the conformation of transmembrane domain 1, the substrate recognition site. These mutants had the opposite modulatory effects of FAD mutations and produced more short Aßs and fewer long Aßs. Our results provide insights into the relationship between PS1 conformational changes and γ-secretase activities.

Compared effects of calcium and sodium polystyrene sulfonate on mineral and bone metabolism and volume overload in pre-dialysis patients with hyperkalemia.

BACKGROUND: Hyperkalemia is prevalent in end-stage renal disease patients, being involved in life-threatening arrhythmias. Although polystyrene sulfonate (PS) is commonly used for the treatment of hyperkalemia, direct comparison of effects between calcium and sodium PS (CPS and SPS) on mineral and bone metabolism has not yet been studied. METHODS: In a randomized and crossover design, 20 pre-dialysis patients with hyperkalemia (>5 mmol/l) received either oral CPS or SPS therapy for 4 weeks. RESULTS: After 4-week treatments, there was no significant difference of changes in serum potassium (K) from the baseline (ΔK) between the two groups. However, SPS significantly decreased serum calcium (Ca) and magnesium (Mg) and increased intact parathyroid hormone (iPTH) values, whereas CPS reduced iPTH. ΔiPTH was inversely correlated with ΔCa and ΔMg (r = -0.53 and r = -0.50, respectively). Furthermore, sodium (Na) and atrial natriuretic peptide (ANP) levels were significantly elevated in patients with SPS, but not with CPS, whereas ΔNa and ΔANP were significantly correlated with each other in all the patients. We also found that ΔNa and Δ(Na to chloride ratio) were positively correlated with ΔHCO3-. In artificial colon fluid, CPS increased Ca and decreased Na. Furthermore, SPS greatly reduced K, Mg, and NH3. CONCLUSION: Compared with SPS, CPS may be safer for the treatment of hyperkalemia in pre-dialysis patients, because it did not induce hyperparathyroidism or volume overload.

Glutamate promotes nucleotide synthesis in the gut and improves availability of soybean meal feed in rainbow trout.

Glutamate (Glu) plays various roles directly or through conversions to other amino acids in intracellular metabolisms such as energy source for enterocytes and precursor for nucleic acids. In this study, we examined the effect of single and chronic oral administration of Glu on cell proliferation in intestine and growth in rainbow trout fed soybean meal (SBM) based diet. In the single dose study, 30, 120 and 360 min after oral administration of 50 and 500 mg/kg Glu, the blood and intestine tissues were collected for amino acid concentration and gene expression analysis. Cell-proliferation was detected 24 h after administration using bromo-deoxy uridine (BrdU) in intestine. In the chronic experiment, fish were fed SBM-based diet added 1 and 2 % of Glu for 8 weeks. Final body weight, plasma amino acid concentrations, gene expression and cell-proliferation in the intestine were analyzed. The expressions of some nucleic acid-synthesis related genes were significantly increased 30 min after administration of 50 mg/kg of Glu. After 8 weeks of feeding, the fish fed SBM-based diet showed significantly lower body weight and microvillus thickness in proximal intestine. Supplementation of 2 % of Glu in the SBM-based feed improved both of them. Though it was not significant difference, Glu tended to increase cell-proliferation in the proximal intestine dose-dependently in both single and chronic administration. Our experiment indicates that Glu has positive effect on rainbow trout fed SBM-based feed by reforming proximal intestine through altering cell-proliferation.

Early nocturnal meal skipping alters the peripheral clock and increases lipogenesis in mice.

BACKGROUND: In humans, skipping meals, especially breakfast, has been associated with obesity and other related syndromes. Recent studies in rodents suggest that fasting and feeding times are potential factors that affect the peripheral circadian clocks and metabolism. However, the link between fasting and obesity in rodents has yet to be fully demonstrated. METHOD: We conducted early nocturnal fasting (ENF) from zeitgeber time (ZT) 12 to 18 for 4 consecutive days in C57B6 mice. The first set of experiments was performed under ad libitum conditions, where ENF and free-feeding (FF) control groups were compared. The second set was performed under isocaloric adjustment by restricting the diet to 90% of the basal intake of ENF mice. Calorie-restricted ENF (ENF-CR) mice were then compared with isocaloric controls (IC-control). Body weight, food intake, core body temperature, activity, adiposity, and clock-related gene expression levels in the liver and adipose tissues were investigated. A stable isotopic analysis was also conducted to estimate de novo lipogenesis fluxes. RESULTS: In the ad libitum condition, the ENF mice ate more during the day, increased their overall daily food intake and gained more weight than FF-control mice. The amplitude of the body core temperature rhythm in ENF mice was also lower than in the FF-controls. Under isocaloric conditions, ENF-CR attenuated the CR-induced body weight loss, compared with the IC-control. ENF-CR also altered the acrophase time of the expression of the clock genes, which is associated with time-shift of genes involved in lipid metabolism and increased lipogenesis, compared with the IC-control. CONCLUSIONS: ENF in nocturnal mice disturbs the peripheral clock and increases de novo lipid synthesis and results in a predisposition to obesity.

A scoring system for histopathologic and immunohistochemical evaluations of uterine leiomyosarcomas.

Uterine leiomyosarcomas (LMS) are difficult to distinguish from benign leiomyomas without surgery. In this study we performed transcervical needle biopsy on 475 patients, 8 LMS patients and 467 patients with non-sarcomas (non-LMS) in a high-risk group for LMS, and evaluated whether examinations performed with Ki-67 and CD34 immunohistochemical analyses in addition to the standard hematoxylin-eosin (H&E)-stained sections would improve preoperative diagnostic precision of the uterine smooth muscle tumors. Histopathologic analysis included three factors: degree of cytologic atypia, mitotic index and coagulative tumor cell necrosis (CTCN). We also evaluated cell proliferation with Ki-67 expression. In cases of suspected CTCN, we examined CD34 expression and counted positive blood vessels in the necrotic area. Three of the 8 LMS cases satisfied the diagnostic criteria of LMS by histopathologic evaluation with H&E-stained sections. We made a score list based on these analyses; scores for LMS specimens ranged from 6-14 points; non-LMS specimens scored 0-2 points. At the cut-off score of 6 points, the positive predictive value to distinguish LMS from non-LMS was 100%, showing that this scoring system, is a useful method for preoperative differentiation between LMS and non-LMS tumors.

Ratio of directly necrotized volume to total volume of a submucosal myoma predicts shrinkage after microwave endometrial ablation.

AIM: To explore parameters relating to shrinkage of submucosal myomas after microwave endometrial ablation. METHODS: Fourteen patients underwent microwave endometrial ablation at 2.45 GHz using a curved microwave applicator to treat menorrhagia caused by a submucosal myoma. The size of myomas was measured on magnetic resonance images before, one month after, and six months after the operation. The average radius r was defined as half of the size of the myoma measured in three dimensions. The protrusion ratio alpha was defined as the ratio of the thickness of the protruding part of a myoma in the uterine cavity to the size of the myoma before the operation. The ratio of the directly necrotized volume by microwave irradiation to the total volume (RODNeV) of preoperative myomas was calculated using the following formula: (1) where d is the depth of myoma tissue directly necrotized by microwave irradiation. The relationships of preoperative alpha, r and RODNeV to postoperative shrinkage were then examined. RESULTS: Two-dimensional plots showing shrinkage of submucosal myomas versus the RODNeV indicated that shrinkage at six months after microwave endometrial ablation depends on the RODNeV. Myomas with a RODNeV greater than 0.17 had shrunk more than 50% at six months after the operation. CONCLUSIONS: RODNeV is closely related to postoperative necrosis and shrinkage of the submucosal myoma after microwave endometrial ablation. Prediction of the shrinkage rate of submucous myomas appears to be possible by calculating the RODNeV preoperatively.

Transcervical microwave myolysis for uterine myomas assisted by transvaginal ultrasonic guidance.

AIM: The effects of transcervical microwave myolysis at 2.45 GHz after microwave endometrial ablation for menorrhagia were examined in patients with myomas. METHODS: A transcervical microwave irradiation system assisted by transvaginal ultrasonic guidance was developed. Ten patients waiting for microwave endometrial ablation for menorrhagia caused by myomas entered the study after complete informed consent was obtained. Their outcomes were examined at least 6 months after transcervical microwave myolysis using the system. After completion of microwave endometrial ablation, interstitial microwave irradiation was performed using a guiding needle set in a puncture adaptor attached to a transvaginal ultrasonic probe and a microwave applicator of 1.6 mm in diameter. Shrinkage of the myoma was measured at 3 months and > or =6 months after the operation. RESULTS: In nine patients with typical myomas, the major part consisting of a submucous or an intramural node 4.0-7.5 cm in size was necrotized. The typical myomas had shrunk by 41-68% at 3 months and 37-69% at > or =6 months after the operation. In one patient with a 6.8-cm cellular leiomyoma, necrosis was limited to the neighborhood of the applicator tip. Shrinkage was 17% at both 3 and 6 months. This patient required a second microwave endometrial ablation at 6 months to treat recurrent menorrhagia. None of the patients underwent hysterectomy after the treatment. No remarkable complications were encountered. CONCLUSIONS: Typical myomas shrink after microwave myolysis following microwave endometrial ablation. Transcervical microwave myolysis seems to be applicable as a low-invasive treatment for a typical myoma.

Expression of glucose transporters in epithelial ovarian carcinoma: correlation with clinical characteristics and tumor angiogenesis.

We immunohistochemically examined the expression of the glucose transporters (GLUT)1, GLUT3 and GLUT4, in 154 tumor samples of epithelial ovarian carcinoma. In addition, we investigated the correlations between the expression of GLUTs and the vascular endothelial growth factor (VEGF), and microvessel count and clinical parameters. The rates of expression of GLUT1, GLUT3 and GLUT4 were 98.7%, 92.8% and 84.4%, respectively. GLUT1 and GLUT4 were both strongly expressed in serous adenocarcinoma, but weakly expressed in clear cell adenocarcinoma. The expressions of GLUT1 and GLUT4 correlated with the clinical disease stage. The expressions of GLUT1, GLUT3 and GLUT4 correlated positively with VEGF expression. The expression status for GLUT1, GLUT3, GLUT4 and VEGF did not represent a prognostic factor. These findings suggest that characteristic differences in the patterns of glucose uptake can exist according to the histological type and that GLUT1, GLUT3 and GLUT4 could be related to tumor angiogenesis in epithelial ovarian carcinoma.