L-theanine elicits an umami taste with inosine 5'-monophosphate

We investigated the taste synergy between L-theanine and the flavour enhancer, inosine 5'-monophosphate (IMP), by using a human sensory evaluation. When L-theanine was added to IMP, only the umami taste was enhanced. We then investigated this synergistic effect of L-theanine in mice by gustatory nerve recording. We confirmed the synergism between L-theanine and IMP for the umami taste.     

TX-2152: a conformationally rigid and electron-rich diyne analogue of FTY720 with in vivo antiangiogenic activity

We designed FTY720 analogues with conformationally rigid and electron-rich acetylenic chains as antiangiogenic agents (the monoyne 1: TX-2148, the diyne 2: TX-2152, the triyne 3: TX-2256). Molecular orbital (MO) calculations of our designed acetylenic analogues and FTY720 showed that the localization of the lowest unoccupied MO and the highest occupied MO increased from phenyl ring to acetylenic chain compared with that of FTY720. These acetylenic analogues were synthesized from p-hydroxyphenylethanol as a starting material. The construction of the acetylenic chain was carried out by an iterative strategy using a Sonogashira cross-coupling reaction and desilylative bromination in two steps. The corresponding overall yields of the monoyne 1, the diyne 2, and the triyne 3 were 27% (11 steps), 13% (13 steps), and 10% (15 steps). The in vivo antiangiogenic activities of these acetylenic analogues and FTY720 were evaluated by the chick embryo chorioallantoic membrane (CAM) assay and compared to the activities of the known antiangiogenic agent TNP-470. The diyne 2 showed more potent antiangiogenic activity (90% inhibition) than FTY720 (77% inhibition) and other acetylenic analogues (the monoyne 1: 42% inhibition, the triyne 3: 60% inhibition), and TNP-470 (82% inhibition) at a dose of 10 microg/CAM, without showing toxicity. The diyne 2 also had potent inhibitory activity at a dose of 5 and 2.5 microg/CAM. These results indicate that the flexibility of C8 alkyl chain of FTY720 is not required for its antiangiogenic activity. We suggest that the diyne 2 (TX-2152) may be a promising candidate as an antiangiogenic agent for antineoplastic drug discovery.     

Structural basis of aspartylglucosaminuria

To elucidate the basis of aspartylglucosaminuria (AGU) from the viewpoint of enzyme structure, we constructed structural models of mutant aspartylglucosaminidase (AGA) proteins using molecular modeling software, TINKER. We classified the amino acid substitutions responsible for AGU and divided them into three groups based on the biochemical phenotype. Then, we examined the structural changes in the AGA protein for each group by calculating the solvent-accessible surface area (ASA), the number of atoms affected, and the root-mean-square deviation (RMSD). Our results revealed that the structural changes in group 1, which exhibits folding/transport defects and a complete deficiency of AGA activity, were generally large and located in the core region of the enzyme molecule. In group 2, exhibiting the mature AGA protein but no AGA activity, the functionally important region of the enzyme molecule was seriously affected. In group 3 exhibiting residual AGA activity, the structural changes in AGA were small and localized near the surface of the enzyme molecule. Coloring of affected atoms based on the distances between the wild-type and mutant ones revealed the characteristic structural changes in the AGA protein geographically and semi-quantitatively. Structural investigation provides us with a deeper insight into the basis of AGU.     

Thyroxine treatment stimulated ovarian follicular angiogenesis in immature hypothyroid rats

The development of mature ovarian follicles is greatly dependent on healthy thecal angiogenesis. Recent experimental evidence showed that thyroxine (T4) treatment promoted ovarian follicle development in immature hypothyroid (rdw) rats. However, an involvement of thyroid hormone in ovarian follicular angiogenesis has not yet been demonstrated. By morphological and molecular approaches, the present studies demonstrated that antral follicles in untreated, T4- or equine chorionic gonadotropin (eCG)-treated rdw rats were mainly small and/or atretic, and presented a poorly developed thecal microvasculature with ultrastructural evidence of diffuse quiescent or degenerative thin capillaries. However, T4 together with eCG increased the number of large antral and mature follicles with numerous activated capillaries and ultra-structural evidence of rich and diffuse angiogenesis in the theca layer. While T4 alone significantly increased mRNA expression of vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNFalpha), it decreased that of fetal liver kinase compared with those in the untreated group. Combined treatment of T4 and eCG markedly increased mRNA abundance of not only VEGF and TNFalpha, but also basic fibroblast growth factor. These data suggest that T4 may promote ovarian follicular angiogenesis in rdw rats by up-regulating mRNA expression of major angiogenic factors.     

Effect of everolimus on the glucose metabolic pathway in mouse skeletal muscle cells (C2C12)

Introduction

Everolimus selectively inhibits mammalian target of rapamycin complex 1 (mTORC1) and exerts an antineoplastic effect. Metabolic disturbance has emerged as a common and unique side effect of everolimus.

Objectives

We used targeted metabolomic analysis to investigate the effects of everolimus on the intracellular glycometabolic pathway.

Methods

Mouse skeletal muscle cells (C2C12) were exposed to everolimus for 48 h, and changes in intracellular metabolites were determined by capillary electrophoresis time-of-flight mass spectrometry. mRNA abundance, protein expression and activity were measured for enzymes involved in glycometabolism and related pathways.

Results

Both extracellular and intracellular glucose levels increased with exposure to everolimus. Most intracellular glycometabolites were decreased by everolimus, including those involved in glycolysis and the pentose phosphate pathway, whereas no changes were observed in the tricarboxylic acid cycle. Everolimus suppressed mRNA expression of enzymes related to glycolysis, downstream of mTOR signaling enzymes and adenosine 5′-monophosphate protein kinases. The activity of key enzymes involved in glycolysis and the pentose phosphate pathway were decreased by everolimus. These results show that everolimus impairs glucose utilization in intracellular metabolism.

Conclusions

The present metabolomic analysis indicates that everolimus impairs glucose metabolism in muscle cells by lowering the activities of glycolysis and the pentose phosphate pathway.

     

Chronic high-fat feeding impairs adaptive induction of mitochondrial fatty acid combustion-associated proteins in brown adipose tissue of mice

Since brown adipose tissue (BAT) is involved in thermogenesis using fatty acids as a fuel, BAT activation is a potential strategy for treating obesity and diabetes. However, whether BAT fatty acid combusting capacity is preserved in these conditions has remained unclear. We therefore evaluated expression levels of fatty acid oxidation-associated enzymes and uncoupling protein 1 (Ucp1) in BAT by western blot using a diet-induced obesity C57BL/6J mouse model. In C57BL/6J mice fed a high-fat diet (HFD) over 2–4 weeks, carnitine palmitoyltransferase 2 (Cpt2), acyl-CoA thioesterase (Acot) 2, Acot11 and Ucp1 levels were significantly increased compared with baseline and control low-fat diet (LFD)-fed mice. Similar results were obtained in other mouse strains, including ddY, ICR and KK-Ay, but the magnitudes of the increase in Ucp1 level were much smaller than in C57BL/6J mice, with decreased Acot11 levels after HFD-feeding. In C57BL/6J mice, increased levels of these mitochondrial proteins declined to near baseline levels after a longer-term HFD-feeding (20 weeks), concurrent with the accumulation of unilocular, large lipid droplets in brown adipocytes. Extramitochondrial Acot11 and acyl-CoA oxidase remained elevated. Treatment of mice with Wy-14,643 also increased these proteins, but was less effective than 4 week-HFD, suggesting that mechanisms other than peroxisome proliferator-activated receptor α were also involved in the upregulation. These results suggest that BAT enhances its fatty acid combusting capacity in response to fat overload, however profound obesity deprives BAT of the responsiveness to fat, possibly via mitochondrial alteration.     

Short term exposure to 1439 MHz pulsed TDMA field does not alter melatonin synthesis in rats

The widespread use of the mobile phone has initiated many studies on the possible adverse effects of a high frequency electromagnetic field (EMF), which is used in mobile phones. A low frequency EMF is reported to suppress melatonin synthesis. The aim of this study was to clarify the effects on melatonin synthesis in rats after short term exposure to a 1439 MHz time division multiple access (TDMA) EMF. The average specific absorption ratio (SAR) of the brain was 7.5 W/kg, and the average SARs of the whole body were 1.9 and 2.0 W/kg for male and female rats, respectively. A total of 208 male and female rats were investigated. After acclimatization to a 12 h light-dark (LD) cycle, serum and pineal melatonin levels together with pineal serotonin level under a dark condition (less than 1 lux) were examined by radioimmunoassay. No significant differences in melatonin and serotonin levels were observed between the exposure, sham, and cage control groups. These results suggest that short term exposure to a 1439 MHz TDMA EMF, which is about four times stronger than that emitted by mobile phones, does not alter melatonin and serotonin synthesis in rats. Further investigations on the effects of long term exposure are warranted.     

Dynamic mechanism for the serpin loop insertion as revealed by quantitative kinetics

The serpin conformational change by insertion of the reactive center loop into beta-sheet A plays a central role in multiple physiological consequences such as serine proteinase inhibition, latency and serpinopathic polymerization. To study the dynamic mechanism for the loop insertion, a novel kinetic method was established utilizing the ovalbumin mutant R339T/A352R; the loop insertion progressed after the cleavage of P1-P1' (Arg352-Ser353) by trypsin was quenched at pH 8 and 0.5 degrees C, and different conformers were quantified by separation using ion-exchange HPLC. The apparent first-order rate constant k(app) determined for various R339T/A352R derivatives differing in conformational stability was greatly increased by lowering the pH. The pH-dependence of k(app) indicated that the protonation of side-chain(s) with a pK(a) value of around 4.6 is a pre-requisite for the loop insertion. The theoretical rate constant k for the protonated form calculated from k(app) was highly variable, depending on the ovalbumin derivative; structural modifications that give increased mobility to helix F and the sheet-A half (s3A/s2A/s1A) resulted in a striking increase in the loop insertion rate constant k. The k values were determined at different temperatures for all the ovalbumin derivatives, and DeltaH(double dagger) and DeltaS(double dagger) values for the loop insertion reaction were determined according to the transition theory. The formation of the transition state was highly endothermic with minor entropy gain, requiring a DeltaG(double dagger) larger than 18 kcal/mol, which can offset the hydrogen-bond cleavages between s3A and s5A. These results are consistent with the transition state with an opened sheet A and altered orientation of helix F.     

Oxidative stability of polyunsaturated fatty acid in phosphatidylcholine liposomes

Synthesized PCs containing docosahexaenoic acid (DHA), arachidonic acid (AA), linoleic acid (LA), and palmitic acid (PA) at known positions in the glycerol moiety were oxidized in liposomes, bulk, and organic solvent. In bulk and organic solvent, the oxidative stability of PC decreased with increasing degrees of unsaturation. However, the degree of unsaturation had little effect on the stability of PC in liposomes. The oxidative stability of PC in liposomes would be affected by the chemical reactivity based on the degree of unsaturation and by the conformation of fatty acyl component in PC bilayers. When the oxidative stability of 1-PA-2-LA-PC or 1-PA-2-AA-PC was compared with that of a 1:1 (mol ratio) mixture of 1,2-diPA-PC + 1,2-diLA-PC, or 1,2-diPA-PC + 1,2-diAA-PC, respectively, the former PC was more oxidatively stable than that of the latter PC mixture in all oxidation systems, although the degree of unsaturation of 1-PA-2-PUFA-PC was the same as that of the corresponding mixture of diPA-PC + diPUFA-PC. The higher oxidative stability of 1-PA-2-PUFA-PC than that of a corresponding mixture of diPA-PC + diPUFA-PC in liposomes was suggested to be due to the different conformation of PC bilayers and the different rate of hydrogen abstraction by free radicals from intermolecular and intramolecular acyl groups.     

Chromosome 4q;10q translocations; Comparison with different ethnic populations and FSHD patients

Background  

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder characterized by the weakness of facial, shoulder-girdle and upper arm muscles. Most patients with FSHD have fewer numbers of tandem repeated 3.3-kb KpnI units on chromosome 4q35. Chromosome 10q26 contains highly homologous KpnI repeats, and inter-chromosomal translocation has been reported.