Acholeplasma vituli sp. nov., from bovine serum and cell cultures.
(73/1766)
Organisms isolated from commercial foetal bovine serum and from cell culture lines containing such serum supplements were found to consist of non-helical, non-motile, pleomorphic coccoid forms. One strain (FC 097-2T) cultivated directly from foetal bovine serum was selected for characterization. In ultrastructural examination, individual round cells lacked cell wall structures and cells varied in size, with a mean diameter of about 700 nm. However, variable numbers of cells were filterable through membranes of 300 nm. Optimum growth occurred between 30 and 37 degrees C. The organism fermented glucose, fructose and mannose, but did not hydrolyse arginine. The strain was insensitive to 500 U penicillin ml(-1) and was capable of growing in the absence of serum or cholesterol. The organism was serologically distinct from all 13 currently described species in the genus Acholeplasma and from other sterol-requiring species in the genus Mycoplasma, using growth inhibition, immunoperoxidase and immunofluorescence tests. Strain FC 097-2T was found to have a DNA G+C composition between 37.6 +/- 1 mol% and 38.3 +/- 1 mol%. The genome size was determined to be 2095 kbp. The 16S rDNA sequence of strain FC 097-2T was compared to 16S rDNA sequences of other mollicutes in nucleotide databases. No deposited sequence was found to be identical; the closest relatives were several members of the genus Acholeplasma. On the basis of these findings and other similarities to acholeplasmas in morphology and growth, the absence of a sterol requirement for growth, and similar genomic characteristics, the organism was assigned to the genus Acholeplasma. Strain FC 097-2T is designated the type strain (ATCC 700667T) of a new species, Acholeplasma vituli. (+info)
Secretory phospholipase A2-potentiated inducible nitric oxide synthase expression by macrophages requires NF-kappa B activation.
(74/1766)
The effect of secretory group II phospholipase A2 (sPLA2) on the expression of the inducible NO synthase (iNOS) and the production of NO by macrophages was investigated. sPLA2 by itself barely stimulated nitrite production and iNOS expression in Raw264.7 cells. However, in combination with LPS, the effects were synergistic. This potentiation was shown for sPLA2 enzymes from sPLA2-transfected stable cells or for purified sPLA2 from human synovial fluid. The effect of PLA2 on iNOS induction appears to be specific for the secretory type of PLA2. LPS-stimulated activation of iNOS was inhibited by the well-known selective inhibitors of sPLA2 such as 12-epi-scalaradial and p-bromophenacyl bromide. In contrast, the cytosolic PLA2-specific inhibitors methyl arachidonyl fluorophosphate and arachidonyltrifluoromethyl ketone did not affect LPS-induced nitrite production and iNOS expression. Moreover, when we transfected cDNA-encoding type II sPLA2, we observed that the sPLA2-transfected cells produced two times more nitrites than the empty vector or cytosolic PLA2-transfected cells. The sPLA2-potentiated iNOS expression was associated with the activation of NF-kappa B. We found that the NF-kappa B inhibitor pyrrolidinedithiocarbamate prevented nitrite production, iNOS induction, and mRNA accumulation by sPLA2 plus LPS in Raw264.7 cells. Furthermore, EMSA analysis of the activation of the NF-kappa B involved in iNOS induction demonstrated that pyrrolidinedithiocarbamate prevented the NF-kappa B binding by sPLA2 plus LPS. Our findings indicated that sPLA2, in the presence of LPS, is a potent activator of macrophages. It stimulates iNOS expression and nitrite production by a mechanism that requires the activation of NF-kappa B. (+info)
Novel processive and nonprocessive glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana synthesize glycoglycerolipids, glycophospholipids, glycosphingolipids and glycosylsterols.
(75/1766)
A processive diacylglycerol glucosyltransferase has recently been identified from Bacillus subtilis [Jorasch, P., Wolter, F.P., Zahringer, U., and Heinz, E. (1998) Mol. Microbiol. 29, 419-430]. Now we report the cloning and characterization of two other genes coding for diacylglycerol glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana; only the S. aureus enzyme shows processivity similar to the B. subtilis enzyme. Both glycosyltransferases characterized in this work show unexpected acceptor specificities. We describe the isolation of the ugt106B1 gene (GenBank accession number Y14370) from the genomic DNA of S. aureus and the ugt81A1 cDNA (GenBank accession number AL031004) from A. thaliana by PCR. After cloning and expression of S. aureus Ugt106B1 in Escherichia coli, SDS/PAGE of total cell extracts showed strong expression of a protein having the predicted size of 44 kDa. Thin-layer chromatographic analysis of the lipids extracted from the transformed E. coli cells revealed several new glycolipids and phosphoglycolipids not present in the controls. These lipids were purified from lipid extracts of E. coli cells expressing the S. aureus gene and identified by NMR and mass spectrometry as 1, 2-diacyl-3-[O-beta-D-glucopyranosyl]-sn-glycerol, 1, 2-diacyl-3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyrano-+ ++syl] -sn-glycerol, 1, 2-diacyl-3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-( 1-->6)-O-beta-D-glucopyranosyl]-sn-glycerol, sn-3'-[O-beta-D-glucopyranosyl]-phosphatidylglycerol and sn-3'-[O-(6"'-O-acyl)-beta-D-glucopyranosyl-(1"'-->6")-O-beta-D-gluco pyranosyl]-sn-2'-acyl-phospha-tidylglycerol. A 1, 2-diacyl-3-[O-beta-D-galactopyranosyl]-sn-glycerol was isolated from extracts of E. coli cells expressing the ugt81A1 cDNA from A. thaliana. The enzymatic activities expected to catalyze the synthesis of these compounds were confirmed by in vitro assays with radioactive substrates. Experiments with several of the above described glycolipids as 14C-labeled sugar acceptors and unlabeled UDP-glucose as glucose donor, suggest that the ugt106B1 gene codes for a processive UDP-glucose:1, 2-diacylglycerol-3-beta-D-glucosyltransferase, whereas ugt81A1 codes for a nonprocessive diacylglycerol galactosyltransferase. As shown in additional assays with different lipophilic acceptors, both enzymes use diacylglycerol and ceramide, but Ugt106B1 also accepts glucosyl ceramide as well as cholesterol and cholesterol glucoside as sugar acceptors. (+info)
Effects of amphotericin B and three azole derivatives on the lipids of yeast cells of Paracoccidioides brasiliensis.
(76/1766)
Yeast cells of five different strains of Paracoccidioides brasiliensis were obtained for partial analysis of lipid composition, and sterol content was determined quantitatively and qualitatively. The determinations were conducted with cells cultured in the presence and absence of amphotericin B and azole derivatives at levels below the MIC. (+info)
Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor.
(77/1766)
Tangier disease, a condition characterized by low levels of high density lipoprotein and cholesterol accumulation in macrophages, is caused by mutations in the ATP-binding cassette transporter ABC1. In cultured macrophages, ABC1 mRNA was induced in an additive fashion by 22(R)-hydroxycholesterol and 9-cis-retinoic acid (9CRA), suggesting induction by nuclear hormone receptors of the liver X receptor (LXR) and retinoid X receptor (RXR) family. We cloned the 5'-end of the human ABC1 transcript from cholesterol-loaded THP1 macrophages. When transfected into RAW macrophages, the upstream promoter was induced 7-fold by 22(R)-hydroxycholesterol, 8-fold by 9CRA, and 37-fold by 9CRA and 22(R)-hydroxycholesterol. Furthermore, promoter activity was increased in a sterol-responsive fashion when cotransfected with LXRalpha/RXR or LXRbeta/RXR. Further experiments identified a direct repeat spaced by four nucleotides (from -70 to -55 base pairs) as a binding site for LXRalpha/RXR or LXRbeta/RXR. Mutations in this element abolished the sterol-mediated activation of the promoter. The results show sterol-dependent transactivation of the ABC1 promoter by LXR/RXR and suggest that small molecule agonists of LXR could be useful drugs to reverse foam cell formation and atherogenesis. (+info)
A critical role of sterols in embryonic patterning and meristem programming revealed by the fackel mutants of Arabidopsis thaliana.
(78/1766)
Here we report a novel Arabidopsis dwarf mutant, fackel-J79, whose adult morphology resembles that of brassinosteroid-deficient mutants but also displays distorted embryos, supernumerary cotyledons, multiple shoot meristems, and stunted roots. We cloned the FACKEL gene and found that it encodes a protein with sequence similarity to both the human sterol reductase family and yeast C-14 sterol reductase and is preferentially expressed in actively growing cells. Biochemical analysis indicates that the fk-J79 mutation results in deficient C-14 sterol reductase activity, abnormal sterol composition, and reduction of brassinosteroids (BRs). Unlike other BR-deficient mutants, the defect of hypocotyl elongation in fk-J79 cannot be corrected by exogenous BRs. The unique phenotypes and sterol composition in fk-J79 indicate crucial roles of sterol regulation and signaling in cell division and cell expansion in embryonic and post-embryonic development in plants. (+info)
Models of luteinization.
(79/1766)
Luteinization is essential to the success of early gestation. It is the process by which elements of the ovarian follicle, usually including both theca interna and granulosa cells, are provoked by the ovulatory stimulus to develop into the corpus luteum. Although there are significant species differences in luteinization, some elements pervade, including the morphological and functional differentiation to produce and secrete progesterone. There is evidence that luteinization results in granulosa cell exit from the cell cycle. The mechanisms that appear to control luteinization include intracellular signalling pathways, cell adhesion factors, intracellular cholesterol and oxysterols, and perhaps progesterone itself as a paracrine or intracrine regulator. Cell models of luteinization, along with some of the conflicting observations on the luteinization process, are discussed in this review. (+info)
Inhibition of sonic hedgehog autoprocessing in cultured mammalian cells by sterol deprivation.
(80/1766)
Sonic hedgehog (Shh) is a signaling molecule that is important for defining patterning in the developing vertebrate central nervous system. After translation, Shh autoproteolyzes and covalently attaches cholesterol to the newly formed carboxyl terminus, a modification crucial for normal Shh signaling. Presented here is evidence that acute severe sterol deprivation in cultured Chinese hamster ovary cells expressing mouse Shh (mShh) inhibits autoprocessing of the protein. These conditions allowed the first detailed kinetic analysis of mShh autoprocessing and turnover rates revealing that cells rapidly degrade both precursor and mature mShh regardless of sterol content and sterol deprivation increases the rate of precursor degradation. Inhibition of mShh autoprocessing also allowed the determination of the subcellular localization of mShh precursor which accumulates in a pre-medial Golgi intracellular compartment. Finally, the precursor form of mShh that results from autoprocessing inhibition appears to accumulate as an amide rather than a stable thioester. (+info)