A G protein-coupled receptor responsive to bile acids

So far some nuclear receptors for bile acids have been identified. However, no cell surface receptor for bile acids has yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile acids specifically induced receptor internalization, the activation of extracellular signal-regulated kinase mitogen-activated protein kinase, the increase of guanosine 5'-O-3-thio-triphosphate binding in membrane fractions, and intracellular cAMP production in Chinese hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5 mRNA showed that it was abundantly expressed in monocytes/macrophages in human and rabbit. Treatment with bile acids was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5 originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not influence those in the original THP-1 cells, suggesting that TGR5 is implicated in the suppression of macrophage functions by bile acids.     

Effect of addition of water-soluble polysaccharides on bacterial cellulose production in a 50-L airlift reactor

Bacterial cellulose (BC) production was carried out in a batch cultivation of Acetobacter xylinum in a 50-L internal loop airlift reactor by addition of water-soluble polysaccharides into the medium. When 0.1% (w/w) agar was added, BC production reached 8.7 g/L compared with 6.3 g/L in the control, and duration of the cultivation period to reach the maximum concentration of BC was almost half of that without addition of polysaccharides. During cultivation, BC was formed into pellets whose size was smaller when the productivity of BC was higher, indicating that increase in the relative viscosity by addition of polysaccharides hindered formation of large clumps of BC and increase in the volumetric oxygen transfer coefficient at high flow rate led to increase in BC productivity.     

Cloning of a gene encoding acetate kinase from Methanosarcina mazei 2-P isolated from a Japanese paddy field soil

The ack gene encoding acetate kinase from the mesophilic Methanosarcina mazei 2-P, isolated from a paddy field soil in Japan, was cloned, sequenced, and functionally expressed in Escherichia coli. The terminal region of the putative pta gene, probably encoding phosphotransacetylase, was found upstream of the ack gene. The deduced amino acid sequence of the acetate kinase is 86.5% identical to that of the Methanosarcina thermophila acetate kinase. The activity of the His(6)-tagged acetate kinase purified from E. coli JM109 was optimal at 35 degrees C.     

Mitochondrial swelling and cytochrome c release: sensitivity to cyclosporin A and calcium

The opening of mitochondrial membrane permeability transition (MPT) pores, which results in a cyclosporin A (CsA)-sensitive and Ca(2+)-dependent dissipation of the membrane potential (delta psi) and swelling (classical MPT), has been postulated to play an important role in the release of cytochrome c (Cyt.c) and also in apoptotic cell death. Recently, it has been reported that CsA-insensitive or Ca(2+)-independent MPT can be classified as non-classic MPT. Therefore, we studied the effects of apoptosis-inducing agents on mitochondrial functions with respect to their CsA-sensitivity and Ca(2+)-dependency. CsA-sensitive mitochondrial swelling, depolarization, and the release of Ca2+ and Cyt.c were induced by low concentrations of arachidonic acid, triiodothyronine (T3), or 6-hydroxdopamine but not by valinomycin and high concentrations of the fatty acid or T3. Fe2+/ADP and 2,2,-azobis-(2-amidinopropane) dihydrochloride (AAPH) induced swelling of mitochondria and the release of Ca2+ and Cyt.c were not coupled with depolarization or CsA-sensitivity while dibucaine-induced swelling occurred without depolarization, Cyt.c-release or by a CsA-sensitive mechanism. A protonophoric FCCP and SF-6847 induced depolarization and Ca(2+)-release occurred in a CsA-insensitive manner and failed to stimulate the release of Cyt.c. These results indicate that ambient conditions of mitochondria can greatly influence the state of membrane stability and that Cyt.c release may occur not only via a CsA-sensitive MPT but also by way of a CsA-insensitive membrane deterioration.     

Mevastatin,an inhibitor of HMG-CoA reductase,induces apoptosis,differentiation and Rap1 expression in HL-60 cells

It has been reported that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase suppress cell proliferation and induce apoptosis. One inhibitor which induces apoptosis is mevastatin. However, the molecular mechanism of apoptosis induction is not well understood so the effects of mevastatin on various functions of HL-60 cells were investigated. We confirmed that mevastatin activated caspase-3 by release of cytochrome c (Cyt. c) from mitochondria through a membrane permeability transition mechanism and also induced typical fragmentation and ladder formation of DNA in HL-60 cells. These effects were inhibited by mevalonate, a metabolic intermediate of cholesterol biosynthesis. Mevalonate and geranylgeraniol (GGOH) inhibited DNA fragmentation whereas farnesol (FOH) did not. Mevastatin also induced cell differentiation to monocytic cells via a mevalonate inhibitable mechanism. Furthermore, mevastatin decreased the amount of an isoprenylated membrane bound Rap1 small GTPase concomitant with an increase in cytosolic Rap1 which occurred before apoptosis and differentiation. On the contrary, both mevastatin and geranylgeranylacetone (GGA), which competes with geranylgeranyl pyrophosphate, induced membrane depolarization of isolated mitochondria without swelling and Cyt. c release. These results suggest that mevastatin-induced apoptosis of HL-60 cells might be caused indirectly by activation of the caspase cascade through the modulation of mitochondrial functions and that some relationship between a certain small GTPase molecule, such as Rap1, and mevastatin-induced apoptosis may exist.     

Chemiluminescence associated with the oxidative metabolism of salicylic acid in rat liver microsomes

Rat liver microsomal suspension (1 mg protein per ml) was incubated at 37 degrees C with 5 mM salicylic acid and 0.2 mM NADPH. The amounts of thiobarbituric acid reactive substances (TBARS) and 2,5-dihydroxybenzoic acid (2,5-DHB), an oxidative metabolite of salicylic acid increased with the incubation time. Simultaneously spontaneous chemiluminescence (CL) was found to be generated there. The addition of SKF-525A, an inhibitor of cytochrome P450 (P450), to the reaction mixture inhibited the CL generation together with the inhibition of the oxidative metabolism. The anti-oxidants and singlet oxygen scavengers like N,N-diphenylphenylenediamine (DPPD) and histidine suppressed the CL generation. The addition of 1,4-diazabicyclo [2.2.2] octane (DABCO), a singlet oxygen quencher, to the reaction mixture generating CL enhanced CL transiently and then CL decreased markedly. Thus CL observed here may possibly originate from the singlet oxygen. The CL generation was suggested to be closely related with salicylic acid-induced lipid peroxidation, and to be coupled with the oxidative metabolism mediated by P450 in rat liver microsomes.     

Rapamycin induces binding activity to the terminal oligopyrimidine tract of ribosomal protein mRNA in rats

Cathepsin G, elastase, and proteinase 3 are serine proteinases released by activated neutrophils. Cathepsin G can cleave angiotensinogen to release angiotensin II, but this activity has not been previously reported for elastase or proteinase 3. In this study we show that elastase and proteinase 3 can release angiotensin I from angiotensinogen and release angiotensin II from angiotensin I and angiotensinogen. The relative order of potency in releasing angiotensin II by the three proteinases at equivalent concentrations is cathepsin G > elastase > proteinase 3. When all three proteinases are used together, the release of angiotensin II is greater than the sum of the release when each proteinase is used individually. Cathepsin G and elastase can also degrade angiotensin II, reactions which might be important in regulating the activity of angiotensin II. The release and degradation of angiotensin II by the neutrophil proteinases are reactions which could play a role in the local inflammatory response and wound healing.     

Intracellular redox regulation by a cystine derivative suppresses UV-induced NF-kappa B activation

Nuclear factor (NF)-kappa B pathways are influenced by the intracellular reduction-oxidation (redox) balance. While NF-kappa B is activated through inhibitor (I)-kappa B degradation by oxidative stress, its DNA binding is accelerated in the reduced state. We found that N,N'-diacetyl-L-cystine dimethylester (DACDM) suppressed the UVB-induced NF-kappa B binding activity at a much lower concentration (50-100 microM) than N-acetyl-L-cysteine (NAC, 10-30 mM). While NAC suppressed the I-kappa B degradation but not the DNA binding, DACDM prevented the activated NF-kappa B from binding DNA, without influencing the I-kappa B degradation. These properties of DACDM make it possible to effectively regulate the intracellular redox balance.     

Effects of pressure on the skin exerted by clothing on responses of urinary catecholamines and cortisol,heart rate and nocturnal urinary melatonin in humans

The study investigated how the pressure exerted on the skin by clothing worn while working in the daytime affected the urinary excretion of adrenaline, noradrenaline and cortisol, heart rate, and also melatonin secretion at night. Nine young women (experiment I) and seven young women (experiment II) participated. Participants wore either a 100% cotton jacket (tight clothes, TC) or a 100% cotton T-shirt (loose clothes, LC). Loose-fitting, 100% cotton tank tops and panties were worn as underwear in both the TC and the LC groups. The main results can be summarized as follows: (1) urinary excretion of adrenaline, noradrenaline and cortisol was facilitated, and the amounts of urinary excretion were significantly higher when TC were worn. Heart rate was significantly higher in the TC group; (2) nocturnal urinary melatonin excretion was significantly greater in the TC group. These results are discussed in terms of an enhancement of diurnal sympathetic nervous system activity caused by pressure on the skin produced by tight clothing.