[Detection of Mycobacterium ulcerans DNA in water bugs collected outside the aquatic environment in Benin]. / Détection des signatures moléculaires de Mycobacterium ulcerans chez des punaises aquatiques capturées au Bénin en dehors de leur environnement aquatique.

Hosting of Mycobacterium ulcerans by water bugs is now well established and their vectoring role has been demonstrated experimentally. These findings were recently corroborated by detection of viable bacilli in the saliva of wild water bugs. However, the extent of water bug involvement in M. ulcerans ecology remains unclear and difficult to evaluate due to lack of understanding about water bug biology. The purpose of this study is to describe the first detection of M. ulcerans DNA in the tissue of water bugs captured outside the aquatic environment. This finding supports the hypothesis that water bug migratory behavior contributes not only to the spread of M. ulcerans but also to transmission outside the aquatic environment.

Contribution of 4-methylthio-2-oxobutanoate and its transaminase to the growth of methionine-dependent cells in culture. Effect of transaminase inhibitors.

The growth in culture of methionine-dependent transformed cells of human, rat and mouse origin was arrested in the absence of L-methionine (Met) but took place in the presence of 4-methylthio-2-oxobutanoic acid (MTOB), the keto acid of Met. From 24 hr after seeding, cells grew in 0.1 mM MTOB medium at a rate comparable to that in 0.1 mM Met medium. Using [35S]MTOB, it was found that the Met synthesized was used in normal MRC-5 cells and in transformed HeLa cells to the same extent for protein, adenosylmethionine and adenosylhomocysteine syntheses. However, when the free Met content was examined, it was found to be 3-fold greater in HeLa than in MRC-5 cells. To examine the importance of this free Met for the growth of transformed cells, the transaminase responsible for converting MTOB to Met was chosen as a target enzyme for the synthesis of compounds with potential inhibitory activity. Since this is a multisubstrate enzyme, reduced Schiff bases were prepared containing both pyridoxal or other aromatic groups, as one constituent, and L-Met or other amino-acids in the free acid or ester or amide form, as the other constituent. Only esters containing the pyridoxal moiety and Met or certain of its structural analogues exhibited good selective growth inhibitory activity in that there was little (20%) or no effect on the growth of normal MRC-5 and derm cells, respectively, while that of transformed HeLa, HEp-2 and L1210 cells was strongly inhibited (80%). This inhibition was accompanied by a concomitant decrease in the activity of the MTOB transaminase in both HeLa and MRC-5 cells treated with 3c the most potent inhibitor. However, using [35S]MTOB it was found that MTOB itself accumulated 48% in HeLa but only 12% in MRC-5 cells treated with 3c. On the contrary [35S]Met formed from [35S]MTOB increased 3.7-fold in MRC-5 inhibitor-treated cells showing 20% growth inhibition whereas it decreased 38% in HeLa-treated cells showing 80% growth inhibition. This decrease in cellular Met in HeLa is not responsible for growth arrest. Indeed the growth of HeLa cells could not be restored by adding a 10-fold excess of Met. Since MTOB can alleviate Met-dependence, the intracellular homeostasis of this metabolite may play a hitherto unsuspected role in controlling cell growth.

Aldehydes from n-6 fatty acid peroxidation. Effects on aminophospholipids.

4-Hydroxy-nonenal (4-HNE) is a major by-product of n-6 fatty acid peroxidation. It has been described to covalently bind biomolecules expressing primary amine, especially the Lys residues in proteins. Low-density lipoproteins (LDL) are well-described macromolecules to be modified by 4-HNE, making them available to scavenger receptors on macrophages. Those macrophages then become foam cells and play an active role in atherogenesis. This paper reports on the covalent binding of 4-HNE to phosphatidylethanolamine (PE), a major aminophospholipid in biological membranes. In contrast, phosphatidylserine (PS) is virtually not modified by 4-HNE. One stable adduct, the Michael adduct PE/4-HNE is a poor substrate of secreted phospholipase A(2) and is not cleaved by phospholipase D. Plasmalogen PE, an important subclass of PE, is covalently modified by 4-HNE as well, but appears to be further degraded on its sn-1 position, the alkenyl chain, which might alter the antioxidant potential of the molecule. An aldehyde homologous to 4-HNE has been characterized as a breakdown product of 12-hydroperoxyeicosatetraenoic acid (12-HpETE) and named 4-hydroxy-2E,6Z-dodecadienal (4-HDDE). This compound as well as 4-HNE was detected in human plasma. Finally, 4-HDDE appears almost 3-fold more active than 4-HNE to make covalent adducts with PE. We conclude that 4-HNE and 4-HDDE are two biologically relevant markers of n-6 fatty acid peroxidation that may alter the phospholipid-dependent cell signaling.

Use of perfluorinated counter ions for the combination of ion pair HPLC and field desorption mass spectrometry. Application to the early characterization of aminoglycoside antibiotics.

The use of perfluorinated carboxylic acids counter ions is described for easy combination of ion pair HPLC and FD-MS. This technique is applied to the analysis of aminoglycoside antibiotics and to the problem of their early characterization from culture broths. Reliable molecular weight informations can be obtained with this method from minute amounts of purified products (10 micrograms) allowing formal identification in most cases.

Synthesis of 9,9,9-trideutero-1,4-dihydroxynonane mercapturic acid (d3-DHN-MA), a useful internal standard for DHN-MA urinalysis.

Racemic 1,4-dihydroxynonane mercapturic acid (DHN-MA) and 9,9,9-trideutero-1,4-dihydroxynonane mercapturic acid (d3-DHN-MA) are synthesized on a 400-mg scale (overall yield approximately 40%) by a two-step sequence involving Michael addition of N-acetyl-L-cysteine to methyl 4-hydroxynon-2(E)-enoate or methyl 9,9,9-trideutero-4-hydroxynon-2 (E)-enoate, followed by reduction of the intermediate adducts with lithium borohydride. The requisite starting methyl esters are obtained, respectively, from heptanal or 7,7,7-trideuteroheptanal and methyl 4-chlorophenylsulfinylacetate via a sulfoxide piperidine and carbonyl reaction described in the literature. The 7,7,7-trideuteroheptanal is easily prepared by classical methods in four steps from 6-bromo-1-hexanol. 13C NMR data indicate that DHN-MA as well as d3-DHN-MA are obtained as mixtures of four diastereomers. Preliminary results show that d3-DHN-MA could be used as an internal standard for mass spectrometric quantification of DHN-MA in human urine.

Synthesis of acetyl,docosahexaenoyl-glycerophosphocholine and its characterization using nuclear magnetic resonance.

Docosahexaenoic acid (DHA) circulates in mammals in lipoproteins and bound to serum albumin as a nonesterified fatty acid as well as esterified in lysophosphatidylcholine (lysoPC). 1-Lyso,2-DHA-glycerophosphocholine (GPC) is an unstable isomer because of a primary alcohol at the sn-1 position. To keep DHA at the sn-2 position of lysoPC, its usual position for the corresponding lysoPC to be acylated into PC in tissues, we synthesized 1-acetyl,2-DHA-GPC and confirmed its structure by use of nuclear magnetic resonance (NMR) spectroscopy in comparison with its positional isomer, 1-DHA,2-acetyl-GPC. 1-Lyso,2-DHA-GPC was prepared from 1-stearoyl,2-DHA-GPC by enzymatic hydrolysis and purified by high-performance liquid chromatography. The isomerization of 1-lyso,2-DHA-GPC into 1-DHA,2-lyso-GPC was obtained by keeping the former overnight at room temperature under nitrogen. Both lysoPC isomers were acetylated by acetic anhydride into 1-acetyl,2-DHA-GPC and 1-DHA,2-acetyl-GPC, respectively, and the resulting phospholipids were fully characterized by NMR. In particular, the 1,2 substitution pattern of the acetyl and DHA chains could be easily detected by 2D heteronuclear multibond correlation. We conclude that 1-acetyl,2-DHA-GPC might be considered as a stable form of 1-lyso,2-DHA-GPC for its delivery to tissues, if the latter exhibits acetyl hydrolase activity.

In vivo and in vitro assessment of UVA protection by sunscreen formulations containing either butyl methoxy dibenzoyl methane, methylene bis-benzotriazolyl tetramethylbutylphenol, or microfine ZnO.

The UVA-attenuating properties of the three UVA filters butyl methoxy dibenzoyl methane (BMDBM), methylene bis-benzotriazolyl tetramethylbutylphenol (MBBT), and microfine zinc oxide (ZnO), are compared. For this purpose persistent pigment darkening (PPD) as an in vivo method as well as different in vitro approaches like the UVA/UVB ratio, the critical wavelength, and the Australian standard have been used. For the case of the UVA/UVB ratio and the critical wavelength the behaviour was also assessed after irradiation with 10 minimal erythemal doses (MED). Sunscreen formulations were manufactured containing either one of these UVA filters or combinations of one UVA filter and a constant amount of UVB filter. The concentration of the respective UVA filter was varied. BMDBM was dissolved in the oil phase of the formulations. MBBT, which is produced as particulate organic UVA-filter dispersion with average particle size smaller than 200 nm, was added to the aqueous phase. The microfine ZnO was incorporated into the oil phase. If no additional UVB filters were present, UVA/UVB ratio and critical wavelength stayed almost constant, independent of UVA-filter content. With constant levels of additional UVB filters these parameters increased with UVA-filter concentration. In contrast to the behaviour of the UVA/UVB ratio, which appeared to be a sensitive measure for the UVA protection in relation to UVB protection in almost the whole ranges of UVA-filter concentrations, the critical wavelength approached saturation already at low UVA-filter levels. The UVA-protection factors (UVA-PF) obtained from the in vivo studies increased with the concentration of the UVA filter in the formulations. Formulations, which showed UVA-PFs > or = 4 in most cases met also the conditions of the Australian Standard. An irradiation dose of 2.5 kJ m(-2) (10 MED) induced significant decreases of UVA/UVB ratio or critical wavelength only with some BMDBM formulations, indicating a loss of UVA protection in those cases.

Specific markers of lipid peroxidation issued from n-3 and n-6 fatty acids.

Several markers of lipid peroxidation are available with different degrees of specificity, from malondialdehyde as a global marker, to F(2)-isoprostane, which is specifically produced from arachidonic acid. Among these, 4-hydroxynonenal is recognized as a breakdown product of fatty acid hydroperoxides, such as 15-hydroperoxy-eicosatetraenoic acid and 13-hydroperoxy-octade cadienoic acid from the n -6 fatty acids. Furthermore, 4-hydroxyhexenal (4-HHE) derives from n -3 fatty acid hydroperoxides. We have recently described the occurrence of 4-hydroxydodecadienal (4-HDDE) from the 12-lipoxygenase product of arachidonic acid 12-hydroperoxy-eicosatetraenoic acid. These three hydroxy-alkenals may be measured in human plasma by GC-MS, but they may partly be generated in the course of sampling, and the relative volatility of 4-HHE makes its measurement quite unreliable. We have successfully characterized and measured the stable oxidized carboxylic acid products from the hydroxy-alkenals 4-HNA, 4-HHA and 4-HDDA in urine. The ratio between 4-HHA and 4-HNA found in the same urinary sample might provide useful information on the location of lipid peroxidation, accounting for the high enrichment of the cerebrovascular system with docosahexaenoic acid, the main n -3 fatty acid in humans.

Altered methional homoeostasis is associated with decreased apoptosis in BAF3 bcl2 murine lymphoid cells.

Methional is a potent inducer of apoptosis in an interleukin 3-dependent murine lymphoid cell line BAF3 b0 when it is added to the culture medium. In these cells transfected with the bcl2 gene, BAF3 bcl2, the apoptotic-inducing activity of methional is dramatically reduced. The addition of disulfiram (an inhibitor of aldehyde dehydrogenase) in order to reduce methional oxidation brought about an increase in apoptosis in BAF3 b0 but not in BAF3 bcl2 cells. In contrast, the addition of quercetin (an inhibitor of aldehyde reductase) in an attempt to diminish methional reduction increased apoptosis in both BAF3 b0 and BAF3 bcl2 cells. The extent of DNA fragmentation in BAF3 bcl2 cells approached that in BAF3 b0 cells in the presence of quercetin and exogenous methional, suggesting a defect in methional biosynthesis in BAF3 bcl2 cells. Direct evidence for this was obtained by measuring labelled methional in cells incubated with the sodium, salt of [U-14C]4-methylthio-2-oxobutanoic acid (MTOB), the precursor of methional. The 80% decrease in labelled methional in BAF3 bcl2 compared with BAF3 b0 cells was accompanied by a concomitant rise in the transamination of [14C]MTOB to [14C]methionine in BAF3 bcl2 cells. Inhibition of the transaminase, however, by a synthetic transition-state-type compound, pyridoxal-L-methionine ethyl ester, induced apoptosis in BAF3 b0 but not in BAF3 bcl2 cells, confirming that the defect in BAF3 bcl2 cells was not in the transaminase itself but rather in the oxidative decarboxylation step MTOB --> methional. In addition, no evidence was obtained for the synthesis of [14C]malondialdehyde from [14C]methional in BAF3 bcl2 cells. As these cells show no deficiency in their content of reactive oxygen species compared with that of BAF3 b0 cells, they may possess some other defect in the beta-hydroxylase enzyme system itself.