Biosynthesis of archaeal membrane lipids: digeranylgeranylglycerophospholipid reductase of the thermoacidophilic archaeon Thermoplasma acidophilum

The basic core structure of archaeal membrane lipids is 2,3-di-O-phytanyl-sn-glyceryl phosphate (archaetidic acid), which is formed by the reduction of 2,3-di-O-geranylgeranylglyceryl phosphate. The reductase activity for the key enzyme in membrane lipid biosynthesis, 2,3-digeranylgeranylglycerophospholipid reductase, was detected in a cell free extract of the thermoacidophilic archaeon Thermoplasma acidophilum. The reduction activity was found in the membrane fraction, and FAD and NADH were required for the activity. The reductase was purified from a cell free extract by ultracentrifugation and four chromatographic steps. The purified enzyme showed a single band at ca. 45 kDa on SDS-PAGE, and catalyzed the formation of archaetidic acid from 2,3-di-O-geranylgeranylglyceryl phosphate. Furthermore, the enzyme also catalyzed the reduction of 2,3-di-O-geranylgeranylglyceryl phosphate analogues such as 2,3-di-O-phytyl-sn-glyceryl phosphate, 3-O-(2,3-di-O-phytyl-sn-glycero-phospho)-sn-glycerol and 2,3-di-O-phytyl-sn-glycero-phosphoethanolamine. The N-terminal 20 amino acid sequence of the purified enzyme was determined and was found to be identical to the sequence encoded by the Ta0516m gene of the T. acidophilum genome. The present study clearly demonstrates that 2,3-digeranylgeranylglycerophospholipid reductase is a membrane associated protein and that the hydrogenation of each double bond of 2,3-digeranylgeranylglycerophospholipids is catalyzed by a single enzyme.     

Potential role for astroglial D-amino acid oxidase in extracellular D-serine metabolism and cytotoxicity

D-amino acid oxidase (DAO) is a flavoenzyme that catalyzes the oxidation of D-amino acids. In the brain, gene expression of DAO is detected in astrocytes. Among the possible substrates of DAO in vivo, D-serine is proposed to be a neuromodulator of the N-methyl-D-aspartate (NMDA) receptor. In a search for the physiological role of DAO in the brain, we investigated the metabolism of extracellular D-serine in glial cells. Here we show that after D-serine treatment, rat primary type-1 astrocytes exhibited increased cell death. In order to enhance the enzyme activity of DAO in cells, we established stable rat C6 glial cells overexpressing mouse DAO designated as C6/DAO. Treatment with a high dose of D-serine led to the production of hydrogen peroxide (H(2)O(2)) followed by apoptosis in C6/DAO cells. Among the amino acids tested, D-serine specifically exhibited a significant cell death-inducing effect. DAO inhibitors, i.e., sodium benzoate and chlorpromazine, partially prevented the death of C6/DAO cells treated with D-serine, indicating the involvement of DAO activity in d-serine metabolism. Overall, we consider that extracellular D-serine can gain access to intracellular DAO, being metabolized to produce H(2)O(2). These results support the proposal that astroglial DAO plays an important role in metabolizing a neuromodulator, D-serine.     

Role of AKT in cyclic strain-induced endothelial cell proliferation and survival

Endothelial cells (ECs) are exposed to repetitive cyclic strain (CS) in vivo by the beating heart. The aim of this study was to assess the influence of CS amplitude and/or frequency on EC proliferation and survival and to determine the role of AKT in CS-induced EC proliferation and survival. Cultured bovine aortic ECs were exposed to 10% strain at a frequency of 60 (60 cpm-10%) or 100 (100 cpm-10%) cycles/min or 15.6% strain at a frequency of 60 cycles/min (60 cpm-15.6%). AKT, glycogen synthase kinase (GSK)-3, BAD, and cleaved caspase-3 were activated by CS in ECs. Increasing the magnitude or frequency of strain resulted in an earlier phosphorylation of GSK-3, although the magnitude of phosphorylation was similar. After CS at 60 cpm-10% for 24 h, the number of nontransfected ECs was significantly increased by 8.5% (P < 0.05). We found that the number of apoptotic ECs was slightly decreased with exposure to CS. ECs transfected with kinase-dead AKT (KA179) as well as plasmids containing a point mutation in the pleckstrin homology domain of AKT (RC25) not only prevented AKT, GSK-3, and BAD phosphorylation but also inhibited the CS-induced increase in cell number as well as the CS-induced protection against apoptosis (both P < 0.05). The ratio of 5'-bromo-2'-deoxyuridine-positive cells was increased when ECs transfected with RC25 and KA179 as well as nontransfected ECs and ECs transfected with Lipofectamine 2000 were exposed to CS. We conclude that AKT is important in enhancing the survival of ECs exposed to CS but is not involved in EC proliferation. apoptosis; glycogen synthase kinase     

Distribution of gamma-hexachlorocyclohexane-degrading genes on three replicons in Sphingobium japonicum UT26

Sphingobium japonicum (formerly Sphingomonas paucimobilis) UT26 utilizes the important insecticide gamma-hexachlorocyclohexane as a sole source of carbon and energy. In previous studies, we isolated and characterized six structural genes (linA to linF) and one regulatory gene (linR) of UT26 for the degradation of gamma-hexachlorocyclohexane to beta-ketoadipate. Our analysis in this study indicated that the UT26 genome consists of three large circular replicons of 3.6 Mb, 670 kb, and 185 kb. The 3.6 Mb and the 670 kb replicons had one and two copies, respectively, of the 16S ribosomal RNA gene, and these replicons were designated as chromosomes (Chr) I and II, respectively. Chr I was indicated to be a main chromosome carrying the dnaA gene. The first three lin genes, linA to linC, for conversion of gamma-hexachlorocyclohexane to 2,5-dichlorohydroquinone, were dispersed on Chr I. The 185 kb plasmid, pCHQ1, carried the linRED operon for the conversion of 2,5-dichlorohydroquinone to maleylacetate and was conjugatively transferred to another sphingomonad strain. The linF gene encoding maleylacetate reductase was located on Chr II. These results indicated that the genes for the complete gamma-hexachlorocyclohexane degradation are dispersed on the three large replicons of UT26.     

Isomer-specific anti-obese and hypolipidemic properties of conjugated linoleic acid in obese OLETF rats

Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers of linoleic acid, has attracted considerable attention because of its potentially beneficial biologic effects both in vitro and in vivo. Our results clearly show the specific action of the 10trans,12cis-CLA isomer against hyperlipidemia and obesity in obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats. After 2 weeks of feeding with 10t,12c-CLA, but not 9cis,11trans-CLA, abdominal adipose tissue weight and serum and hepatic lipid levels in OLETF rats were lower than those in linoleic acid-fed rats. These effects were attributable to suppressed fatty acid synthesis and enhanced fatty acid beta oxidation in the liver on a 10t,12c-CLA diet. Additionally, we showed that mRNA expression of fatty acid synthase, carnitine palmitoyltransferase, leptin, and sterol regulatory element binding protein-1 was also regulated by 10t,12c-CLA. We suppose that 10t,12c-CLA reveals hypolipidemic and anti-obese activity through the alteration of mRNA expressions in the liver and white adipose tissue.     

Comprehensive multigene phylogenies of excavate protists reveal the evolutionary positions of "primitive" eukaryotes

Many of the protists thought to represent the deepest branches on the eukaryotic tree are assigned to a loose assemblage called the "excavates." This includes the mitochondrion-lacking diplomonads and parabasalids (e.g., Giardia and Trichomonas) and the jakobids (e.g., Reclinomonas). We report the first multigene phylogenetic analyses to include a comprehensive sampling of excavate groups (six nuclear-encoded protein-coding genes, nine of the 10 recognized excavate groups). Excavates coalesce into three clades with relatively strong maximum likelihood bootstrap support. Only the phylogenetic position of Malawimonas is uncertain. Diplomonads, parabasalids, and the free-living amitochondriate protist Carpediemonas are closely related to each other. Two other amitochondriate excavates, oxymonads and Trimastix, form the second monophyletic group. The third group is comprised of Euglenozoa (e.g., trypanosomes), Heterolobosea, and jakobids. Unexpectedly, jakobids appear to be specifically related to Heterolobosea. This tree topology calls into question the concept of Discicristata as a supergroup of eukaryotes united by discoidal mitochondrial cristae and makes it implausible that jakobids represent an independent early-diverging eukaryotic lineage. The close jakobids-Heterolobosea-Euglenozoa connection demands complex evolutionary scenarios to explain the transition between the presumed ancestral bacterial-type mitochondrial RNA polymerase found in jakobids and the phage-type protein in other eukaryotic lineages, including Euglenozoa and Heterolobosea.     

The Group B Streptococcus NADH oxidase Nox-2 is involved in fatty acid biosynthesis during aerobic growth and contributes to virulence

Numerous Streptococcaceae produce an H2O-forming NADH oxidase, Nox-2, which has been generally implicated in aerobic survival. We examined the roles of Nox-2 in Group B Streptococcus (GBS), a leading agent of neonatal infections. While nox2 inactivation caused an aerobic growth arrest, no improvement was seen by addition of antioxidants to cultures, suggesting that this defect was not due to accumulation of toxic oxygen species. Using several approaches, we show that the observed inability of the nox2 mutant to grow aerobically is mainly due to an underlying defect in fatty acid (FA) biosynthesis: (i) the nox2 aerobic growth defect is fully and rapidly complemented by adding oleic acid to culture medium, and (ii) direct assimilation of this unsaturated FA in both wild type (WT) and nox2 GBS membranes is demonstrated and correlated with mutant growth rescue. We propose that NAD+ depletion in the nox2 mutant results in reduced acetyl-CoA production, which perturbs FA biosynthesis and hence blocks growth in aerobiosis. The nox2 aerobic growth defect was also complemented when GBS respiration metabolism was activated by exogenous haem and menaquinone. The membrane NADH oxidase activity generated by the functional respiratory chain thus compensates the cytoplasmic NADH oxidase deficiency. The nox2 mutant was attenuated for virulence, as assessed in lung, intraperitoneal and intravenous murine infection models. As the nox2 defect seems only to affect aerobic growth of GBS, its reduced virulence supports the suggestion that aerobic conditions and NADH oxidase activities are relevant to the GBS infection process.     

Influence of vitamin E on gastric mucosal injury induced by Helicobacter pylori infection

We investigated the effect of vitamin E on gastric mucosal injury induced by Helicobacter pylori (H. pylori) infection. Male Mongolian gerbils were divided into 4 groups (normal group without H. pylori infection, vitamin E-deficient, -sufficient and -supplemented groups with H. pylori infection). Following oral inoculation with H. pylori (ATCC43504 2 x 10(8) CFU), animals were fed diets alpha-tocopherol 2 mg/100 g diet in the normal and vitamin E-sufficient groups and alpha-tocopherol 0.1 mg/100 g and 50 mg/100 g in the vitamin E-deficient and -supplemented groups, respectively, for 24 weeks. Chronic gastritis was detected in all gerbils inoculated H. pylori. Gastric ulcer was detected in 2 of 7 gerbils only in the vitamin E-deficient group. In the vitamin E-deficient group, myeloperoxidase activity and mouse keratinocyte derived chemokine (KC) in gastric mucosa was significantly higher than in the vitamin E supplemented group. Subsequently, in an in vitro study expression of CD11b/CD18 on neutrophils was enhanced by H. pylori water extract. This effect was suppressed in a dose dependent manner by the addition of alpha-tocopherol. These results suggest that vitamin E has a protective effect on gastric mucosal injury induced by H. pylori infection in gerbils, through the inhibition of accumulation of activated neutrophils.     

Differential effect of DHEA on mitogen-induced proliferation of T and B lymphocytes

Dehydroepiandrosterone (DHEA) is the predominant steroid hormone secreted by adrenal gland, and it has been proposed in recent years that DHEA has significant effects on immune function. We investigated the effect of DHEA (1 x 10(-5) - 1 x 10(-8)M) on proliferation of human T cells and B cells and on immunoglobulin production, a representative function of B cells. High doses of DHEA (1 x 10(-5)) significantly inhibited proliferation of peripheral blood mononuclear cells (PBMCs) and T cells induced by T cell mitogens hemagglutinin (PHA) and concanavalin A (Con A). Proliferation of PBMCs induced by B cell mitogens pokeweed mitogen (PWM) was increased by 1 x 10(-7) - 1 x 10(-6)M DHEA. Proliferation of PBMCs and B cells induced by Staphylococcus aureus Cowan strain I (SAC) was not significantly changed at any concentrations of DHEA. However, a concentration of 1 x 10(-7)M DHEA tended to potentiate their proliferation. This study suggested that DHEA acted on T and B lymphocytes differentially in immune system.     

CYP707A3, a major ABA 8'-hydroxylase involved in dehydration and rehydration response in Arabidopsis thaliana

Abscisic acid (ABA) catabolism is one of the determinants of endogenous ABA levels affecting numerous aspects of plant growth and abiotic stress responses. The major ABA catabolic pathway is triggered by ABA 8'-hydroxylation catalysed by the cytochrome P450 CYP707A family. Among four members of Arabidopsis CYP707As, the expression of CYP707A3 was most highly induced in response to both dehydration and subsequent rehydration. A T-DNA insertional cyp707a3-1 mutant contained higher ABA levels in turgid plants, which showed a reduced transpiration rate and hypersensitivity to exogenous ABA during early seedling growth. On dehydration, the cyp707a3-1 mutant accumulated a higher amount of stress-induced ABA than the wild type, an event that occurred relatively later and was coincident with slow drought induction of CYP707A3. The cyp707a3 mutant plants exhibited both exaggerated ABA-inducible gene expression and enhanced drought tolerance. Conversely, constitutive expression of CYP707A3 relieved growth retardation by ABA, increased transpiration, and a reduction of endogenous ABA in both turgid and dehydrated plants. Taken together, our results indicate that CYP707A3 plays an important role in determining threshold levels of ABA during dehydration and after rehydration.