Methyl palmitate inhibits lipopolysaccharide-stimulated phagocytic activity of rat peritoneal macrophages

Macrophages, in general, are critical effectors of body's immune system. Chemical inhibition of phagocytic activity of such macrophages as Kupffer cells has been extensively studied. We have earlier shown that methyl palmitate (MP) inhibits the activation of Kupffer cells. To evaluate the potential of MP to inhibit the activation of other macrophages, we treated rat peritoneal macrophages with varying concentrations of MP. Its treatment led to a dose-dependent inhibition of phagocytic activity, which was found to be 34%, 47%, and 66% at 0.25, 0.50, and 1.0 mM MP, respectively, as measured by latex bead uptake. When MP-treated peritoneal macrophages were stimulated with lipopolysaccharide (LPS), the nitric oxide (.NO) release was inhibited at 6 h, while cyclooxygenase-2 expression decreased after 24 h. The treatment with MP increased the release of interleukin (IL)-10 in the LPS-treated cells at 6 h, while IL-6 and tumor necrosis factor-alpha were significantly increased both at 6 and 24 h. Our data suggest that MP inhibits phagocytic activity and .NO production similar to that observed in isolated Kupffer cells. Therefore, inhibition of phagocytosis by MP may be a general phenomenon, and it could be used as an inhibitor of macrophage function.     

Precursor limitations in methyl jasmonate-induced Catharanthus roseus cell cultures

Jasmonates enhance the expression of various genes involved in terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus. We applied precursor feeding to our C. roseus suspensions to determine how methyl jasmonate (MJ) alters the precursor availability for TIA biosynthesis. C. roseus suspensions were induced with MJ (100 μM) on day 6 and fed loganin (0.30 mM), tryptamine (0.15 mM), loganin plus tryptamine, or geraniol (0.1–1.0 mM) on day 7. While MJ increased ajmalicine production by 3-fold, induced cultures were still limited by terpenoid precursors. However, both induced and non-induced cultures became tryptamine-limited with excess loganin. Geraniol feeding also increased ajmalicine production in non-induced cultures. But MJ appeared to increase geraniol availability in induced cultures, due presumably to the increased expression of Dxs with MJ addition.     

dUTP Pyrophosphatase, its appearance in extracellular compartment may serve as a potential biomarker for N-methyl-N'-nitro-N-nitrosoguanidine exposure in mammalian cells

The monofunctional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a model chemical widely used for studying the molecular events induced by the widespread environmental N-nitroso alkylating carcinogen. Many studies have focused on understanding MNNG-induced mutagenesis and carcinogenesis. However, the search for specific indicators of MNNG exposure is still underway. In this study, we analyzed the proteins in culture medium of human amnion epithelial cells (FL cells) exposed to MNNG by 2-DE followed by MALDI-TOF MS, in the hope of finding a specific protein marker suitable for MNNG risk assessment. Image visualization and statistical analysis indicated that 12 spots appeared and 4 spots up-regulated after MNNG exposure. Most of them were identified by MS. These proteins include nuclear isoform of dUTP pyrophosphatase (DUT-N), phosphoglycerate mutase 1, heparan sulfate proteoglycan perlecan, etc., which are involved in multiple cellular functions. Interestingly, 2-DE and MS analyses of cell lysate exposed to MNNG revealed that DUT-N was down-regulated. The appearance of DUT-N in culture medium and its down-regulation in cell lysate was confirmed by Western blot. These data suggest that these proteins, especially DUT-N, could be used as candidate biomarkers for monitoring MNNG exposure.     

Comparative study on methyl orange removal by growing cells and washed cell suspensions of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> TISTR 1500

Lactobacillus casei TISTR 1500 possesses cytoplasmic azoreductase, and converts methyl orange to N,N-dimethyl-p-phenylenediamine and 4-aminobenzenesulfonic acid. In culture growth, the strain completely degraded methyl orange at 200 mg/l, even though the pH value was lower than 4. The decolorization was inhibited in the growing culture with 800 mg of the dye/l after incubation for 12 h. The percentage of decolorization and specific decolorization rate with 400 and 800 mg/l were 66 and 15%, and 14.2, and 8.7 mg/gCell/h, respectively. Additionally, a growing culture is more tolerant to a high initial dye concentration than when using washed cell suspensions supplied with only sucrose. Moreover, incubation of a low cell density in 600 μM of Na⁺ and 20 mM of sucrose increased the specific decolorization rate from 2.34 mg/gCell/h (without Na⁺) to 4.32 mg/gCell/h. However, Na⁺ had no effect on the enhancement of azoreductase activity in the reaction mixture.     

Khayanolides from African mahogany Khaya senegalensis (Meliaceae): A revision

Five khayanolides (1-O-acetylkhayanolide B 1, khayanolide B 2, khayanolide E 3, 1-O-deacetylkhayanolide E 4, 6-dehydroxylkhayanolide E 5) were isolated from the stem bark of African mahogany Khaya senegalensis (Meliaceae). Their structures and absolute configurations were determined through extensive spectroscopic analyses including MS, NMR, and single-crystal X-ray diffraction experiments. The results established that two previously reported khayanolides, 1α-acetoxy-2β,3α,6,8α,14β-pentahydroxy-[4.2.1^10,30.1^1,4]-tricyclomeliac-7-oate 6 and 1α,2β,3α,6,8α,14β-hexahydroxy-[4.2.1^10,30.1^1,4]-tricyclomeliac-7-oate 7, were, in fact, 1-O-acetylkhayanolide B 1 and khayanolide B 2, and that the two reported phragmalin derivatives, methyl 1α-acetoxy-6,8α,14β,30β-tetrahydroxy-3-oxo-[3.3.1^10,2.1^1,4]-tricyclomeliac-7-oate 8 and methyl 1α,6,8α,14β,30β-pentahydroxy-3-oxo-[3.3.1^10,2.1^1,4]-tricyclomeliac-7-oate 9, were, in fact, khayanolide E 3 and 1-O-deacetylkhayanolide E 4, respectively. Based on the results from this study and consideration of the biogenetic pathway, the methyl 6-hydroxyangolensate in African mahogany K. senegalensis should have a C-6 S configuration while methyl 6-hydroxyangolensate in genuine mahogany Swietenia species should have a C-6 R configuration.     

Optimization of the transient transformation of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cells by particle bombardment and its application to the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase and geraniol 10-hydroxylase

The monoterpene indole alkaloids (MIA) synthesized in Catharanthus roseus are highly valuable metabolites due to their pharmacological properties. In planta, the MIA biosynthetic pathway exhibits a complex compartmentation at the cellular level, whereas subcellular data are sparse. To gain insight into this level of organization, we have developed a high efficiency green fluorescent protein (GFP) imaging approach to systematically localize MIA biosynthetic enzymes within C. roseus cells following a biolistic-mediated transient transformation. The biolistic transformation protocol has been first optimized to obtain a high number of transiently transformed cells with a ~12-fold increase compared to previous protocols and thus to clearly and easily identify the fusion GFP expression patterns in numerous cells. On the basis of this protocol, the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase (HDS), a methyl erythritol phosphate pathway enzyme and geraniol 10-hydroxylase (G10H), a monoterpene-secoiridoid pathway enzyme has been next characterized. Besides showing the accumulation of HDS within plastids of C. roseus cells, we also provide evidences of the presence of HDS in long stroma-filled thylakoid-free extensions budding from plastids, i.e. stromules that are in close association with other organelles such as endoplasmic reticulum (ER) or mitochondria in agreement with their proposed function in enhancing interorganelle metabolite exchanges. Furthermore, we also demonstrated that G10H is an ER-anchored protein, consistent with the presence of a transmembrane helix at the G10H N-terminal end, which is both necessary and sufficient to drive the ER anchoring. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mineralization of methyl tert-butyl ether and other gasoline oxygenates by Pseudomonads using short n-alkanes as growth source

Biodegradation of methyl tert-butyl ether (MTBE) by cometabolism has shown to produce recalcitrant metabolic intermediates that often accumulate. In this work, a consortium containing Pseudomonads was studied for its ability to fully degrade oxygenates by cometabolism. This consortium mineralized MTBE and TBA with C3-C7 n-alkanes. The highest degradation rates for MTBE (75 +/- 5 mg g(protein) (-1) h(-1)) and TBA (86.9 +/- 7.3 mg g(protein) (-1) h(-1)) were obtained with n-pentane and n-propane, respectively. When incubated with radiolabeled MTBE and n-pentane, it converted more than 96% of the added MTBE to (14)C-CO(2). Furthermore, the consortium degraded tert-amyl methyl ether, tert-butyl alcohol (TBA), tert-amyl alcohol, ethyl tert-butyl ether (ETBE) when n-pentane was used as growth source. Three Pseudomonads were isolated but only two showed independent MTBE degradation activity. The maximum degradation rates were 101 and 182 mg g(protein) (-1) h(-1) for Pseudomonas aeruginosa and Pseudomonas citronellolis, respectively. The highest specific affinity (a degrees (MTBE)) value of 4.39 l g(protein) (-1) h(-1) was obtained for Pseudomonas aeruginosa and complete mineralization was attained with a MTBE: n-pentane ratio (w/w) of 0.7. This is the first time that Pseudomonads have been reported to fully mineralize MTBE by cometabolic degradation.     

Novel synthesis of methyl 4,6-O-benzylidenespiro[2-deoxy-α-d-arabino-hexopyranoside-2,2′-imidazolidine] and its homologue and sugar-γ-butyrolactam derivatives from methyl 4,6-O-benzylidene-α-d-arabino-hexopyranosid-2-ulose

Novel methyl 4,6-O-benzylidenespiro[2-deoxy-α-d-arabino-hexopyranoside-2,2′-imidazolidine] and its homologue methyl 4,6-O-benzylidene-3′,4′,5′,6′-tetrahydro-1′H-spiro[2-deoxy-α-d-arabino-hexopyranoside-2,2′-pyrimidine] have been synthesized in good yields by reaction of methyl 4,6-O-benzylidene-α-d-arabino-hexopyranosid-2-ulose with 1,2-diaminoethane and 1,3-diaminopropane. The results are completely different from the reaction with arylamines or alkylamines. One-pot synthesis of novel (E)-methyl 4-[hydroxy (methoxy)methylene]-5-oxo-1-alkyl-(4,6-O-benzylidene-2-deoxy-α-d-glucopyranosido)[3,2-b]pyrrolidines has been achieved by the reaction of alkylamines with the butenolide-containing sugar, derived from the aldol condensation of methyl 4,6-O-benzylidene-α-d-arabino-hexopyranosid-2-ulose with diethyl malonate. These sugar-γ-butyrolactam derivatives are potential GABA receptor ligands.     

Metal-mediated allylation of enzymatically oxidized methyl α-D-galactopyranoside

The C-6 unit of methyl α-d-galactopyranoside was selectively modified by combining enzymatic oxidation with an indium-mediated allylation reaction. The Barbier-Grignard type reaction, where a carbonyl group reacts with an allyl halide, proceeds in aqueous solution, even with water as the only solvent; thus carbohydrates can be modified without the need for drying or protection-deprotection steps. The corresponding homoallyl alcohols are produced in high yields of >90% in the reactions with allyl bromide and cinnamyl chloride. The main products were isolated and characterized by GC-MS and NMR spectroscopy.