Omega-hydroxy Fatty Acids Research Articles

Deposition and localization of lipid polyester in developing seeds of Brassica napus and Arabidopsis thaliana

Mature seeds of Arabidopsis thaliana and Brassica napus contain complex mixtures of aliphatic monomers derived from non-extractable lipid polyesters. Most of the monomers are deposited in the seed coat, and their compositions suggest the presence of both cutin and suberin layers. The location of these polyesters within the seed coat, and their contributions to permeability of the seed coat and other functional properties are unknown. Polyester deposition was followed over Brassica seed development and distinct temporal patterns of monomer accumulation were observed. Octadecadiene-1,18-dioate, the major leaf cutin monomer, was transiently deposited. In contrast, the saturated dicarboxylates maintained a constant level during seed desiccation, whereas the fatty alcohols and saturated omega-hydroxy fatty acids continually increased. Dissection and analysis of Brassica seed coats showed that suberization is not specific to the chalaza. Analysis of the Arabidopsis ap2-7 mutant suggested that suberin monomers are preferentially associated with the outer integument. Several Arabidopsis knockout mutant lines for genes involved in polyester biosynthesis (att1, fatB and gpat5) were examined for seed monomer load and composition. The variance in polyester monomers of these mutants is correlated with dye penetration assays. Furthermore, stable transgenic plants expressing promoter::YFP fusions showed ATT1 promoter activity in the inner integument, whereas GPAT5 promoter is active in the outer integument. Together, the Arabidopsis data indicated that there is a suberized layer associated with the outer integument and a cutin-like polyester layer associated with the inner seed coat.

The acyltransferase GPAT5 is required for the synthesis of suberin in seed coat and root of Arabidopsis.

Suberin and cutin are fatty acid- and glycerol-based plant polymers that act as pathogen barriers and function in the control of water and solute transport. However, despite important physiological roles, their biosynthetic pathways, including the acyl transfer reactions, remain hypothetical. We report the characterization of two suberin mutants (gpat5-1 and gpat5-2) of Arabidopsis thaliana GPAT5, encoding a protein with acyl-CoA:glycerol-3-phosphate acyltransferase activity. RT-PCR and beta-glucuronidase-promoter fusion analyses demonstrated GPAT5 expression in seed coat, root, hypocotyl, and anther. The gpat5 plants showed a 50% decrease in aliphatic suberin in young roots and produced seed coats with a severalfold reduction in very long chain dicarboxylic acid and omega-hydroxy fatty acids typical of suberin but no change in the composition or content of membrane or storage glycerolipids or surface waxes. Consistent with their altered suberin, seed coats of gpat5 mutants had a steep increase in permeability to tetrazolium salts compared with wild-type seed coats. Furthermore, the germination rate of gpat5 seeds under high salt was reduced, and gpat5 seedlings had lower tolerance to salt stress. These results provide evidence for a critical role of GPAT5 in polyester biogenesis in seed coats and roots and for the importance of lipid polymer structures in the normal function of these organs.

Molecular Cloning of CYP76B9, a Cytochrome P450 fromPetunia hybrida, Catalyzing the ω-Hydroxylation of Capric Acid and Lauric Acid

A cDNA encoding a cytochrome P450 (CYP76B9) was isolated from Petunia hybrida. Northern blot analysis revealed preferential expression of the gene in flowers and leaves. The recombinant yeast microsomes expressing CYP76B9 was allowed to react with capric acid and lauric acid as substrates. One major metabolite was produced from each fatty acid after incubation with yeast microsomes expressing CYP76B9. The metabolites were identified by gas chromatography-mass spectrometry (GC-MS) as omega-hydroxy capric acid and omega-hydroxy lauric acid. The kinetic parameters of the reactions were Km=9.4 microM and Vmax=13.6 mol min(-1) per mol of P450 for capric acid, and Km=5.7 microM and Vmax=19.1 mol min(-1) per mol of P450 for lauric acid. We found that the omega-hydroxy metabolites of capric acid and lauric acid can affect the plant growth of Arabidopsis thaliana. Plants grown in the presence of omega-hydroxy fatty acids exhibited shorter root length than control plants with the corresponding non-hydroxylated fatty acids.

The outermost cuticle of soybean seeds: chemical composition and function during imbibition

Seeds of different cultivars of Glycine max (L.) Merr. (soybean) have strikingly different rates of water imbibition. Seeds that readily imbibe water are termed 'soft', while those that remain non-permeable, even after several days in water, are referred to as 'hard', 'stone', or 'impermeable' seeds. What prevents soybean hard seeds from taking up water? Previous work established that the initial imbibition of soft soybean seeds correlates with the presence of small cracks in the outermost cuticle that covers the seed coat, prompting a detailed analysis of soybean seed coat cutin. In this paper, it is shown that the outermost cuticle of the seed coat has an unusual chemical composition, lacking typical mid-chain-hydroxylated fatty acids but being relatively rich in other types of hydroxylated fatty acids. The cuticle of the impermeable cultivar studied contained a disproportionately high amount of hydroxylated fatty acids relative to that of the permeable ones. Moreover, a brief treatment with hot alkali released the omega-hydroxy fatty acid component of the outermost cuticle and created holes in it that caused the seeds to become permeable. This demonstrates that the outermost cuticle of the seed is the critical structure that prevents water uptake by hard seeds.

Depletion of ceramides with very long chain fatty acids causes defective skin permeability barrier function, and neonatal lethality in ELOVL4 deficient mice

Very long chain fatty acids (VLCFA), either free or as components of glycerolipids and sphingolipids, are present in many organs. Elongation of very long chain fatty acids-4 (ELOVL4) belongs to a family of 6 members of putative fatty acid elongases that are involved in the formation of VLCFA. Mutations in ELOVL4 were found to be responsible for an autosomal dominant form of Stargardt's-like macular dystrophy (STGD3) in human. We have previously disrupted the mouse Elovl4 gene, and found that Elovl4+/- mice were developmentally normal, suggesting that haploinsufficiency of ELOVL4 is not a cause for the juvenile retinal degeneration in STGD3 patients. However, Elovl4-/- mice died within several hours of birth for unknown reason(s). To study functions of ELOVL4 further, we have explored the causes for the postnatal lethality in Elovl4-/- mice. Our data indicated that the mutant mice exhibited reduced thickness of the dermis, delayed differentiation of keratinocytes, and abnormal structure of the stratum corneum. We showed that all Elovl4-/- mice exhibited defective skin water permeability barrier function, leading to the early postnatal death. We further showed that the absence of ELOVL4 results in depletion in the epidermis of ceramides with omega-hydroxy very long chain fatty acids (> or = C28) and accumulation of ceramides with non omega-hydroxy fatty acids of C26, implicating C26 fatty acids as possible substrates of ELOVL4. These data demonstrate that ELOVL4 is required for VLCFA synthesis that is essential for water permeability barrier function of skin.

Chemical Composition of Abaca (Musa textilis) Leaf Fibers Used for Manufacturing of High Quality Paper Pulps

The chemical composition of leaf fibers of abaca (Musa textilis), which are commonly used for high-quality paper pulp production, was thoroughly studied. The results revealed that the lignin content was 13.2% of the total fiber. The analysis of abaca fibers by pyrolysis coupled to gas chromatography-mass spectrometry (Py-GC/MS) released predominantly compounds arising from lignin and p-hydroxycinnamic acids, with high amounts of 4-vinylphenol. The latter compound was demonstrated to arise from p-coumaric acid by pyrolysis of abaca fibers in the presence of tetramethylammonium hydroxide, which released high amounts of p-coumaric acid (as the methyl derivative). Products from p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) propanoid units, with a predominance of the latter (H:G:S molar ratio of 1.5:1:4.9), were also released after Py-GC/MS of abaca fibers. Sinapyl and coniferyl acetates, which are thought to be lignin monomer precursors, were also found in abaca. The extractives content of the abaca fiber (0.4%) was low, and the most predominant compounds were free sterols (24% of total extract) and fatty acids (24% of total extract). Additionally, significant amounts of steroid ketones (10%), triglycerides (6%), omega-hydroxyfatty acids (6%), monoglycerides (4%), fatty alcohols (4%), and a series of p-hydroxycinnamyl (p-coumaric and ferulic acids) esterified with long chain alcohols and omega-hydroxyfatty acids were also found, together with minor amounts of steroid hydrocarbons, diglycerides, alpha-hydroxyfatty acids, sterol esters, and sterol glycosides.

Lipids as renewable resources: current state of chemical and biotechnological conversion and diversification

Oils and fats are the most important renewable raw materials of the chemical industry. They make available fatty acids in such purity that they may be used for chemical conversions and for the synthesis of chemically pure compounds. Oleic acid (1) from "new sunflower," linoleic acid (2) from soybean, linolenic acid (3) from linseed, erucic acid (4) from rape seed, and ricinoleic acid (5) from castor oil are most important for chemical transformations offering in addition to the carboxy group one or more C-C-double bonds. New plant oils containing fatty acids with new and interesting functionalities such as petroselinic acid (6) from Coriandrum sativum, calendic acid (7) from Calendula officinalis, alpha-eleostearic acid (8) from tung oil, santalbic acid (9) from Santalum album (Linn.), and vernolic acid (10) from Vernonia galamensis are becoming industrially available. The basic oleochemicals are free fatty acids, methyl esters, fatty alcohols, and fatty amines as well as glycerol as a by-product. Their interesting new industrial applications are the usage as environmentally friendly industrial fluids and lubricants, insulating fluid for electric utilities such as transformers and additive to asphalt. Modern methods of synthetic organic chemistry including enzymatic and microbial transformations were applied extensively to fatty compounds for the selective functionalization of the alkyl chain. Syntheses of long-chain diacids, omega-hydroxy fatty acids, and omega-unsaturated fatty acids as base chemicals derived from vegetable oils were developed. Interesting applications were opened by the epoxidation of C-C-double bonds giving the possibility of photochemically initiated cationic curing and access to polyetherpolyols. Enantiomerically pure fatty acids as part of the chiral pool of nature can be used for the synthesis of nonracemic building blocks.

ω-Oxidation of Very Long-chain Fatty Acids in Human Liver Microsomes: IMPLICATIONS FOR X-LINKED ADRENOLEUKODYSTROPHY

X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder biochemically characterized by elevated levels of very long-chain fatty acids (VLCFA). Excess levels of VLCFAs are thought to play an important role in the pathogenesis of X-ALD. Therefore, therapeutic approaches for X-ALD are focused on the reduction or normalization of VLCFAs. In this study, we investigated an alternative oxidation route for VLCFAs, namely omega-oxidation. The results described in this study show that VLCFAs are substrates for the omega-oxidation system in human liver microsomes. Moreover, VLCFAs were not only converted into omega-hydroxy fatty acids, but they were also further oxidized to dicarboxylic acids via cytochrome P450-mediated reactions. High sensitivity toward the specific P450 inhibitor 17-octadecynoic acid suggested that omega-hydroxylation of VLCFAs is catalyzed by P450 enzymes belonging to the CYP4A/F subfamilies. Studies with individually expressed human recombinant P450 enzymes revealed that two P450 enzymes, i.e. CYP4F2 and CYP4F3B, participate in the omega-hydroxylation of VLCFAs. Both enzymes belong to the cytochrome P450 4F subfamily and have a high affinity for VLCFAs. In summary, this study demonstrates that VLCFAs are substrates for the human omega-oxidation system, and for this reason, stimulation of the in vivo VLCFA omega-oxidation pathway may provide an alternative mode of treatment to reduce the levels of VLCFAs in patients with X-ALD.

Analysis of the aliphatic monomer composition of polyesters associated with Arabidopsis epidermis: occurrence of octadeca‐cis‐6, cis‐9‐diene‐1,18‐dioate as the major component

Although the surface waxes from Arabidopsis thaliana leaves and stems have been thoroughly characterized, the monomer composition of the polyesters of the cuticular membrane has not been analyzed. Delipidated Arabidopsis leaves or stems, when depolymerized under conditions to cleave polyesters, produced typical omega-hydroxy fatty acid cutin monomers such as 16-hydroxy-palmitate, 10,16-dihydroxy-palmitate and 18-hydroxy-9,10-epoxy-stearate. However, the major monomer was octadeca-cis-6, cis-9-diene-1,18-dioate, with lesser amounts of octadec-cis-9-ene-1,18-dioate and hexadeca-1,16-dioate. These dicarboxylates were found predominantly in epidermal peels from Arabidopsis stems and are therefore likely to be associated with the cuticular membrane. They were also found in analyses of canola leaves but were absent in tomato and apple fruit cutins. In the fad2-1 mutant line of Arabidopsis, which has reduced levels of linoleate and linolenate and elevated oleate in cytosolic phospholipids, the amount of octadeca-cis-6, cis-9-diene-1,18-dioate was 50% reduced, with a concomitant increase in octadec-cis-9-ene-1,18-dioate. In a fatb-ko line of Arabidopsis, where the availability of cytosolic palmitate is impaired, there was an 80% loss of C16 monomers and a compensating increase in C18 monomers. The presence of substantial amounts of dicarboxylates in cuticular membranes is unexpected. High amounts of aliphatic dicarboxylates are usually considered as an indicator of suberin, and are reported only as very minor components of cutin. The high level of polyunsaturation is also unusual in cuticles; saturated fatty acid monomers usually predominate, with lesser amounts of monounsaturates. These novel findings for Arabidopsis demonstrate that a broad range of monomer compositions are possible for polyesters of the epidermis.

Identification of intact long-chain p-hydroxycinnamate esters in leaf fibers of abaca (Musa textilis) using gas chromatography/mass spectrometry.

The study of acetone-extractable components from the leaf fibers of the non-wood plant abaca (Musa textilis) resulted in the isolation and identification of series of intact hydroxycinnamate esters consisting of ferulic and p-coumaric acids esterified to long-chain fatty alcohols (C20 to C28) and omega-hydroxyfatty acids (C22 to C28). These series of compounds were characterized by high-temperature gas chromatography/mass spectrometry (GC/MS) using capillary columns (12 m length) with thin films that allowed the analysis of intact (i.e., without prior saponification) hydroxycinnamate esters. Characterization of intact individual compounds was achieved based on the mass spectra obtained by GC/MS of the underivatized compounds and their methyl and/or trimethylsilyl ether derivatives.

Enhancement of carcinostatic activity of ω-hydroxy fatty acids by their esterification through increased uptake into tumor cells

Diverse omega-hydroxy fatty acids (omegaHFAs) and their derivatives were examined for their ability to diminish the cell viability of Ehrlich ascites tumor cells by the mitochondrial dehydrogenase-based WST-1 assay and trypan blue dye exclusion assay. Of the diverse omegaHFAs, hydroxyhexadecanoic acid (omegaH16:0) was appreciably carcinostatic, and hydroxypentadecanoic acid (omegaH15:0) or hydroxypentadecenoic acid (omegaH15:1) was weakly carcinostatic at a dose of 100 micro M, whereas hydroxydodecanoic acid (omegaH12:0) and hydroxyhexadecenoic acid (omegaH16:1) acid were scarcely carcinostatic at the same dose. In contrast their sodium salt derivatives except omegaH16:0 were not carcinostatic. Enhancement of the carcinostatic activity was markedly exerted by ethylesterization of omegaHFAs with the saturated fatty moiety such as omegaH16:0 and omegaH15:0, whereas ethylesters of the unsaturated omegaHFAs such as omegaH15:1 and omegaH16:1 were weakly carcinostatic. Thus intramolecular introduction of a double bond was shown to weaken the carcinostatic activity in case of either omegaHFAs or their ethylester, being in contrast to the conventional knowledge concerning the enhancement of carcinostatic activities of non-hydroxy fatty acids appendant with more double bonds. The intracellular uptake amount of each omegaHFA as quantified by gas chromatography was the following order: omegaH16:0 ethylester (10.1 pg/cell) > omegaH15:0 ethylester (6.4 pg/cell) > omegaH16:0 (3.4 pg/cell) > omegaH15:0 (2.8 pg/cell), which accords with the order of carcinostatic activities of four saturated omegaHFAs in contrast to discord between both the orders for unsaturated omegaHFAs which could be scarcely detected as the intact form within cells. The results indicate that enhancement of carcinostatic activity of omegaH16:0 by ethylesterization was attributed to an appreciable correlation between intracellular uptake amounts and carcinostatic activities for diverse omegaHFAs with saturated fatty moiety, being not true for unsaturated omegaHFAs.

Transformation of fatty acids catalyzed by cytochrome P450 monooxygenase enzymes of Candida tropicalis.

Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in beta-oxidation convert these substrates to long-chain alpha,omega-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is omega-hydroxylation by a cytochrome P450 (CYP) monooxygenase. C. tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C(18:1)), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C. tropicalis cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to omega-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C(14:0)) much more effectively. Both enzymes, in particular CYP52A17, also oxidized omega-hydroxy fatty acids, ultimately generating the alpha,omega-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for beta-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.

Deficiency of Epidermal Protein-Bound ω-Hydroxyceramides in Atopic Dermatitis

Atopic dermatitis is a common skin disease of unknown etiology with an impaired permeability barrier function. To learn more about the molecular pathology in lesional skin, we analyzed levels of free extractable as well as protein-bound barrier lipids in the epidermis of atopic dermatitis subjects. The amount of protein-bound omega-hydroxyceramides in healthy epidermis comprised 46-53 wt% of total protein-bound lipids, whereas this percentage was decreased to 23-28 wt% in nonlesional areas and even down to 10-25 wt% in affected atopic skin areas of the subjects. Furthermore, the partial amount of free extractable very long chain fatty acids with more than 24 carbon atoms was reduced in affected regions down to 25 wt% and in nonlesional regions of the atopic dermatitis subjects down to 40 wt% compared to healthy controls. This "hydrocarbon chain length deficiency" regarding the barrier lipids in atopic skin was supported by metabolic labeling studies with [14C]-serine in cultured epidermis. The biosynthesis of free glucosylceramides and free ceramides was remarkably decreased in affected skin areas of the atopic subjects compared to healthy control subjects. Especially affected were the de novo syntheses of ceramide 4 (i.e., ceramide EOH, consisting of a very long chain N-acyl omega-hydroxy fatty acid esterified with linoleic acid and 6-hydroxysphingosine as sphingoid base) and ceramide 3 (ceramide NP, consisting of a nonhydroxy N-acyl fatty acid and phytosphingosine). In conclusion, this study revealed that the lesional epidermis in atopic dermatitis has considerable deficiencies within main barrier lipid components, which may contribute to the severely damaged permeability barrier.

Identification of omega hydroxy fatty acids in biological samples as their pentafluoropropyl derivatives by gas chromatography/mass spectrometry with positive and negative ion detection

A simple one-step procedure for derivatization of the omega hydroxy fatty acids 20-hydroxyeicotetraeonic acid and 12-hydroxylauric acid is presented. The procedure involves acylation of the terminal hydroxy group and esterification of the carboxylic acid with a mixture of pentafluoropropionic anhydride and pentafluoropropanol. Positive and negative ion spectra for the derivatives are presented. The procedure was used to demonstrate conversion of arachidonic acid to 20-hydroxyeicosatetraeonic acid and lauric acid to 12-hydroxylauric acid in kidney microsomal incubations. The reaction appears to be specific, since derivatives of subterminal fatty acids (secondary alcohols) could not be detected.

The thermotropic phase behaviour of phyto-ceramide 1 as investigated by ATR-FTIR and DSC

Phyto-ceramide 1 {N-(27-stearoyloxy-heptacosanoyl)-phytosphingosine} consists of a C18 phytosphingosine base coupled via an amide link to a very long chain omega-hydroxy fatty acid, which is linked to a further fatty acid. This unusual ceramide found in the lipid barrier of stratum corneum is thought to have important functions for the integrity of the permeability barrier in the stratum corneum. Phyto-ceramide 1 functions as a molecular rivet holding together different bilayers of the lipid barrier. In addition, it is assumed that phyto-ceramide 1 interacts with the corneocyte cell membranes of the stratum corneum and thus fixes these cells to the stratum corneum lipid matrix. The thermotropic phase behaviour of phyto-ceramide 1 was investigated by means of attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and differential scanning calorimetry (DSC). The aim of this study was the understanding of the organisation of phyto-ceramide 1 bilayers and their thermotropic phase behaviour. Special attention was focused on the intermolecular hydrogen bonding interactions in the polar interface and with water molecules during lipid film hydration. From the calorimetric data the phase transition temperature was determined to 105°C with a very large phase transition enthalpy ΔH of +146 kJ mol−1. FTIR provided information on the hydrophobic core of the lipids as well as information concerning head group interactions. Analysing the methylene stretching, scissoring and rocking mode indicated a highly ordered gel phase. Hydration of the lipid films had some impact on the reorganisation of the acyl chains as is derived from the band shape and frequencies of the amide I and II bands. Water penetration into the polar lipid region was monitored by the temperature dependence of the amide II and O–H/N–H stretching intensities as a function of H→D exchange. Hydration could only be realized at the onset of the phase transition, which indicated that strong intermolecular interactions between the ceramide head groups exist. Hydration of the lipid film induced the formation of a tighter hydrogen bonding network between the ceramide head groups, as concluded from the position of both amide bands. The analysis of the band of the ester carbonyl group located in the “middle” of the ceramide structure revealed that it is involved in hydrogen bonding. This was concluded from the changes of the ester carbonyl band upon heating and hydration.

Barrier Characteristics of Different Human Skin Types Investigated with X-Ray Diffraction, Lipid Analysis, and Electron Microscopy Imaging

The stratum corneum requires ceramides, cholesterol, and fatty acids to provide the cutaneous permeability barrier. The lipids are organized in intercellular membranes exhibiting short- and long-periodicity lamellar phases. In recent years, the phase behavior of barrier lipid mixtures has been studied in vitro. The relationship of human stratum corneum lipid composition to membrane organization in vivo, however, has not been clearly established. Furthermore, the special function of the different ceramide species in the stratum corneum is largely unknown. We examined lipid organization and composition of stratum corneum sheets from different subtypes of healthy human skin (normal, dry, and aged skin). Lipid organization was investigated using X-ray diffraction and demonstrated that the 4.4 nm peak attributed to the long periodicity phase was frequently missing for skin with a low Cer(EOS)/Cer(total) ratio, indicating an important part for Cer(EOS), which contains omega-hydroxy fatty acid (O) ester-linked to linoleic acid (E) and amide-linked to sphingosine (S). A deficiency in the 4. 4 nm peak was predominantly observed in young dry skin. In one case of aged skin, however, and less often in young normal skin this peak was also missing. Furthermore, the ceramide composition of samples without the 4.4 nm peak showed a deficiency of Cer(EOH), which contains 6-hydroxy-4-sphingenine (H), and an increase in Cer(NS) and Cer(AS), which contain nonhydroxy (N) or alpha-hydroxy fatty acids (A). In addition, a 3.4 nm peak attributed to crystalline cholesterol occurred in most cases of aged and dry skin, but was not observed in young normal skin. Our results do not indicate a definite pattern of correlation between lipid organization and types of human skin. They demonstrate, however, that Cer(EOS) and Cer(EOH) are key elements for the molecular organization of the long periodicity lamellar phase in the human stratum corneum.

Two Ceramide Subfractions Detectable in Cer(AS) Position by HPTLC in Skin Surface Lipids of Non-Lesional Skin of Atopic Eczema

The non-involved skin of atopic eczema (NEAE) is characterized by severe dryness and an impaired barrier function of the stratum corneum as indicated by an increased transepidermal water loss. Previous studies have demonstrated that this barrier impairment coincides with marked alterations in the amount and composition of stratum corneum ceramides. The aim of this study was to identify specific alterations in NEAE that may be used in the diagnosis of the atopic eczema. Using a classical procedure for high performance thin layer chromatography we could confirm earlier results: apart from Cer(EOH), which contains omega-hydroxy fatty acid (O) ester-linked to linoleic acid (E) and amide-linked to 6-hydroxy-4-sphingenine (H), the quantities of all ceramide fractions were significantly decreased. Furthermore, Cer(EOH)/Certotal was significantly increased, whereas the percentage of Cer(EOS), which contains sphingosine (S), and Cer(NP), which contains non-hydroxy fatty acid (N) amide-linked to phytosphingosine (P), were significantly decreased. Using a modified procedure for high performance thin layer chromatography we could demonstrate the formation of a double peak in the position of Cer(AS), which contains alpha-hydroxy fatty acid (A), in lipids of NEAE. The subfractions of the double peak comprised 15% and 12% of Certotal. MALDITOF mass spectrometry suggested that the double peak was formed by a homologous series of mono-hydroxylated and mono-unsaturated ceramides of different chain length, e.g., Cer(AS) subfractions containing either (C16,18) or (C22,24,26) alpha-hydroxy fatty acids. In contrast, in normal skin a single peak in Cer(AS) position, which comprised 22% of Certotal, was mainly formed by the long chain subfraction. In some cases this single peak displayed a small shoulder at its right flank, but never showed a clear peak separation when developed with NEAE samples. Furthermore, even in senile xerosis, or in either non-involved skin of psoriasis or seborrhoic eczema, only a single peak occurred in Cer(AS) position. Accordingly, the double peak might be specific for NEAE and turn out to be a marker for atopic eczema.

A Novel Subtype of Class II Alcohol Dehydrogenase in Rodents: UNIQUE PRO47 and SER182 MODULATES HYDRIDE TRANSFER IN THE MOUSE ENZYME

Mice and rats were found to possess class II alcohol dehydrogenases with novel enzymatic and structural properties. A cDNA was isolated from mouse liver and the encoded alcohol dehydrogenase showed high identity (93.1%) with the rat class II alcohol dehydrogenase which stands in contrast to the pronounced overall variability of the class II line. The two heterologously expressed rodent class II enzymes exhibited over 100-fold lower catalytic efficiency (k(cat)/K(m)) for oxidation of alcohols as compared with other alcohol dehydrogenases and were not saturated with ethanol. Hydride transfer limited the rate of octanol oxidation as indicated by a deuterium isotope effect of 4.8. The mutation P47H improved hydride transfer and turnover rates were increased to the same level as for the human class II enzyme. Michaelis constants for alcohols and aldehydes were decreased while they were increased for the coenzyme. The rodent class II enzymes catalyzed reduction of p-benzoquinone with about the same maximal turnover as for the human form. This activity was not affected by the P47H mutation while a S182T mutation increased the K(m) value for benzoquinone 10-fold. omega-Hydroxy fatty acids were catalyzed extremely slow but functioned as potent inhibitors by binding to the enzyme-NAD(+) complex. All these data indicate that the mammalian class II alcohol dehydrogenase line is divided into two structurally and functionally distinct subgroups.

Increase production of lactonizing lipase (LipL) from Pseudomonas sp. strain 109 by lipids and detergents.

LipL of Pseudomonas sp. strain 109 is a unique lipase capable of catalyzing macrocyclic lactone synthesis using omega-hydroxyfatty acid esters as substrates. Several fatty acid esters were tested as inducers of LipL production. The addition of either soybean oil or a non-ionic detergent (Noigen HC) resulted in a 44 to 45-fold increase in extracellular LipL, and the presence of both resulted in a further 56-fold increase. Among the triglycerides tested, triolein was the most effective, with a 50-fold increase in LipL production. A Northern blot hybridization analysis found that the lipL transcript increased in the presence of soybean oil or Noigen HC, indicating that the production of LipL is regulated at the transcriptional level.

Emission of Climate-Relevant Trace Gases and Succession of Microbial Communities during Open-Windrow Composting

Determination of different indicators of microbial biomass, community structure, and bioactivity by the fumigation extraction method, as well as determination of phospholipid fatty acids (PLFAs) and their subfractions and the measurement of trace gases, respectively, provides valuable information about microbial succession in composting processes. The emission rates of carbon dioxide (CO(inf2)), methane (CH(inf4)), and nitrous oxide (N(inf2)O) increased successively during compost maturation: initially in the presence of easily degradable nutrients, during high temperature, and after the temperature had cooled down, respectively. The emission rate patterns of these trace gases corresponded to the concentrations of PLFAs and their particular subfractions. (i) Similar to the CO(inf2) emission rates, microbial biomass estimations by fumigation extraction and by determination of the amount of total PLFAs showed a discontinuous decrease during the composting process, with a slight increase at the end of the observation period. (ii) An increase in ether lipids, indicating the enhanced presence of archaean methanogens, and an elevated CH(inf4) emission were observed at the same time. (iii) The period of enhanced N(inf2)O emission corresponded to the increase in beta and omega hydroxy fatty acids derived from the outer membrane. Additionally, the continuous increase in branched-chain fatty acids suggested an increase in gram-positive bacteria and actinomycetes, and the decrease in polyunsaturated fatty acids indicated a decrease in eukaryotic cells during the composting.