Discovery of novel (4-piperidinyl)-piperazines as potent and orally active acetyl-CoA carboxylase 1/2 non-selective inhibitors: F-Boc and triF-Boc groups are acid-stable bioisosteres for the Boc group

Novel (4-piperidinyl)-piperazine derivatives were synthesized and evaluated as ACC1/2 non-selective inhibitors. Optimization of the substituents on the nitrogen of the piperidine ring led to the identification of the fluorine substituted tert-butoxycarbonyl group. Advanced analog, 1,1,1-trifluoro-2-methylpropan-2-yl 4-{4-[(2-amino-6-methyl-1-benzothiophen-3-yl)carbonyl]piperazin-1-yl}piperidine-1-carboxylate (12c) showed potent inhibitory activities in enzyme-assay and cell-based assays. Compound 12c also exhibited reduction of hepatic de novo fatty acid synthesis in rats after oral administration.     

The composition of newly synthesized proteins in the endoplasmic reticulum determines the transport pathways of soybean seed storage proteins

Glycinin (11S) and beta-conglycinin (7S) are major storage proteins in soybean (Glycine max L.) seeds and accumulate in the protein storage vacuole (PSV). These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the PSV by vesicles. Electron microscopic analysis of developing soybean cotyledons of the wild type and mutants with storage protein composition different from that of the wild type showed that there are two transport pathways: one is via the Golgi and the other bypasses it. Golgi-derived vesicles were observed in all lines used in this study and formed smooth dense bodies with a diameter of 0.5 to several micrometers. ER-derived protein bodies (PBs) with a diameter of 0.3-0.5 microm were observed at high frequency in the mutants containing higher amount of 11S group I subunit than the wild type, whereas they were hardly observed in the mutants lacking 11S group I subunit. These indicate that pro11S group I may affect the formation of PBs. Thus, the composition of newly synthesized proteins in the ER is important in the selection of the transport pathways.     

Effect of unsaturated bonds in the sn-2 acyl chain of phosphatidylcholine on the membrane-damaging action of Clostridium perfringens alpha-toxin toward liposomes

Clostridium perfringens alpha-toxin degrades phosphatidylcholine (PC) in the bilayer of liposomes and destroys the membrane. The effect of the type and position of unsaturation in the fatty acyl chain of PC (18:0/18:1 PC) synthesized on the toxin-induced leakage of carboxyfluorescein (CF) from PC liposomes was examined. Differential scanning calorimetry showed that the phase transition temperature (T(m)) was minimal when the triple bond was positioned at C (9) in the sn-2 acyl chain. The toxin-induced CF leakage decreased with the migration of the bond from C (9) to either end of the acyl chain in PC. The PC containing the cis-double bond had a similar T(m) to that with the triple bond, but a lower value than the PC containing the trans-double bond. Furthermore, the toxin-induced leakage from liposomes composed of PC containing the cis-double bond resembled that with PC having the triple bond and was greater than that from liposomes with PC having the trans-double bond. The binding of a H148G mutant to PC liposomes showed a reciprocal relationship in terms of the T(m) value of PC containing the triple bond. These results indicate that the toxin-induced membrane damage is closely related to membrane fluidity in liposomes.     

Enhancing effect of dietary vinegar on the intestinal absorption of calcium in ovariectomized rats.

We studied the effect of dietary vinegar on calcium absorption by using ovariectomized rats fed on a low-calcium diet. The apparent absorption of calcium was higher when the rats were fed on a diet containing 1.6% vinegar for 32 days than when fed on a diet without vinegar (P < 0.05). The calcium content in the femur of the rats given diets containing 0.4% and 1.6% vinegar were also higher (P < 0.05). The serum parathyroid hormone level was lower and the crypt depth of the duodenum thicker in the rats fed on a diet containing 1.6% vinegar (P < 0.05). These results suggest that dietary vinegar enhanced intestinal calcium absorption by improving calcium solubility and by the trophic effect of the acetic acid contained in vinegar, which would reduce the bone turnover caused by ovariectomy and be helpful in preventing osteoporosis.     

Physiologically high concentrations of 17beta-estradiol enhance NF-kappaB activity in human T cells

Estrogen has diverse effects on inflammation and immune responses. That pregnancy is associated with remission of some autoimmune diseases and exacerbation of others suggests that physiological fluctuation in estrogen levels could affect the immune responses in humans. However, the molecular basis for these phenomena is poorly understood. We hypothesized that fluctuations of estrogen levels modulate intracellular signaling for immune responses via estrogen receptors (ERs). In reporter assays, 17beta-estradiol (E2) at a physiologically high concentration increased the activity of NF-kappaB in Jurkat cells stimulated by PMA/ionomycin or TNF-alpha. Overexpression and RNA interference experiments suggested that the effects were mediated through ERbeta. Immunoprecipitation assay showed that both ERalpha and ERbeta are directly associated with NF-kappaB in the cell nucleus. Using chromatin immunoprecipitation assay, we confirmed that ERalpha and ERbeta associated with NF-kappaB and steroid hormone coactivators at the promoter region of NF-kappaB regulated gene. Considering that NF-kappaB regulates the expression of various genes essential for cell growth and death, estrogen could regulate the fate of T cells by affecting the activity of NF-kappaB. To determine whether E2 alters the fate of T cells, we investigated E2 actions on T cell apoptosis, a well-known NF-kappaB-mediated phenomenon. E2 increased apoptosis of Jurkat cells and decreased that of human peripheral blood T cells. Our results indicate that E2 at a physiologically high concentration modulates NF-kappaB signaling in human T cells via ERbeta and affects T cell survival, suggesting that these actions may underlie the gender differences in autoimmune diseases.     

The role of heavy-metal ATPases,HMAs, in zinc and cadmium transport in rice

The P_1B-type heavy metal ATPases (HMAs) are diverse in terms of tissue distribution, subcellular localization, and metal specificity. Functional studies of HMAs have shown that these transporters can be divided into two subgroups based on their metal-substrate specificity: a copper (Cu)/silver (Ag) group and a zinc (Zn)/cobalt (Co)/cadmium (Cd)/lead (Pb) group. Studies on Arabidopsis thaliana and metal hyperaccumulator plants indicate that HMAs play an important role in the translocation or detoxification of Zn and Cd in plants. Rice possesses nine HMA genes, of which OsHMA1–OsHMA3 belong to the Zn/Co/Cd/Pb subgroup. OsHMA2 plays an important role in root-to-shoot translocation of Zn and Cd, and participates in Zn and Cd transport to developing seeds in rice. OsHMA3 transports Cd and plays a role in the sequestration of Cd into vacuoles in root cells. Modification of the expression of these genes might be an effective approach for reducing the Cd concentration in rice grains.     

The catalytic architecture of leukotriene C4 synthase with two arginine residues

Leukotriene (LT) C(4) and its metabolites, LTD(4) and LTE(4), are involved in the pathobiology of bronchial asthma. LTC(4) synthase is the nuclear membrane-embedded enzyme responsible for LTC(4) biosynthesis, catalyzing the conjugation of two substrates that have considerably different water solubility; that amphipathic LTA(4) as a derivative of arachidonic acid and a water-soluble glutathione (GSH). A previous crystal structure revealed important details of GSH binding and implied a GSH activating function for Arg-104. In addition, Arg-31 was also proposed to participate in the catalysis based on the putative LTA(4) binding model. In this study enzymatic assay with mutant enzymes demonstrates that Arg-104 is required for the binding and activation of GSH and that Arg-31 is needed for catalysis probably by activating the epoxide group of LTA(4).     

Biosynthetic pathway of phytosiderophores in iron-deficient graminaceous plants: A new assay system for the detection of nicotianamine amino-transferase activity

A new assay system for the detection of nicotianamine amino-transferase activity was developed. The activity of nicotianamine amino-transferase which participated in biosynthetic pathway of MAs from methionine in graminaceous plants was induced by the iron deficiency treatment.