Immunocytochemical localization of Δ3, Δ2-enoyl-CoA isomerase and NADPH-dependent-2,4-dienoyl-CoA reductase in rat kidney

Localization of ³, ²-enoyl-CoA isomerase (ECI) and NADPH-dependent-2,4-dienoyl-CoA reductase (DCR) in the rat kidney was investigated by immunocytochemical techniques. The kidneys were perfusion-fixed and embedded in Epon or LR White. For light microscopy, semi-thin sections of Epon-embedded materials were stained by the immunoenzyme technique after the epoxy resin was removed by treatment with sodium ethoxide. For electron microscopy, ultra-thin sections of LR White-embedded materials were stained by the protein A-gold technique. By light microscopy, the S1 segment of the proximal tubule was most heavily stained for ECI and DCR whilst S2 and S3 segments showed intermediate staining. A weak staining reaction was observed in the distal tubule and the medullary collecting tubule. In the cortical collecting tubule, heavily stained cells were present between weakly stained cells. By electron microscopy, gold particles showing the antigenic sites for ECI were confined mainly to the mitochondria, but few particles were observed in the peroxisomes. Gold labeling for DCR was localized both in the mitochondria and the peroxisomes. The labeling intensity of the peroxisomes was much higher than that of the mitochondria. The results suggest that metabolism of unsaturated fatty acids occurs mainly in the mitochondria and the peroxisomes of the proximal tubule in the kidney.     

Putative function and physiological relevance of the mitochondrial uncoupling protein-3: involvement in fatty acid metabolism?

The discovery of the human homologue of the thermogenic protein UCP1, named uncoupling protein 3 (UCP3), boosted research on the role of this skeletal muscle protein in energy metabolism and body weight regulation. Nowadays, 9 years after its discovery emerging data indicate that the primary physiological role of UCP3 may be the mitochondrial handling of fatty acids rather than regulating energy expenditure via thermogenesis. UCP3 has been proposed to export fatty acid anions or fatty acid peroxides away from the matrix-side of the mitochondrial inner membrane to prevent their deleterious accumulation. In this way, UCP3 could protect mitochondria against lipid-induced oxidative mitochondrial damage, a function especially important under conditions of high fatty acid supply to skeletal muscle mitochondria. Such function may be clinically relevant in the development of type 2 diabetes mellitus, a condition characterized by muscular fat accumulation, mitochondrial damage and low levels of UCP3.     

Characterization of a monoclinic crystal form of an enzyme of steroid metabolism, Δ5-3-ketosteroid isomerase

A monoclinic crystal form ($\text{P2}{}_1\text{, a = 140.4}\text{A}\text{?}\text{, b = 85.0}\text{A}\text{?}\text{, c = 94.5}\text{A}\text{?}$, β= 130.1 °) of Δ⁵-3-ketosteroid isomerase from Pseudomonas testosteroni (EC 5.3.3.1), grown at pH 7.0, has been characterized. Crystal-density measurements show that the asymmetric unit contains 12 protomers (M_r = 13,394).     

Gene identification and functional characterization of a Δ12 fatty acid desaturase in Tetrahymena thermophila and its influence in homeoviscous adaptation to low temperature

Homeoviscous adaptation in poikilotherms is based in the regulation of the level of desaturation of fatty acids, variation in phospholipids head groups and sterol content in the membrane lipids, in order to maintain the membrane fluidity in response to changes in environmental temperature. Increased proportion of unsaturated fatty acids is thought to be the main response to low-temperature acclimation, which is mostly achieved by fatty acid desaturases. Genome analysis of the ciliate Tetrahymena thermophila and a gene knockout approach has allowed us to identify one Δ12 FAD and to study its activity in the original host and in a yeast heterologous expression system. The “PUFA index” -relative content of polyunsaturated fatty acids compared to the sum of saturated and monounsaturated fatty acid content- was ~57% lower at 15 °C and 35 °C in the Δ12 FAD gene knockout strain (KOΔ12) compared to WT strain. We characterized the role of T. thermophila Δ12 FAD on homeoviscous adaptation and analyzed its involvement in cellular growth, cold stress response, and membrane fluidity, as well as its expression pattern during temperature shifts. Although these alterations allowed normal growth in the KOΔ12 strain at 30 °C or higher temperatures, growth was impaired at temperatures of 20 °C or lower, where homeoviscous adaptation is impaired. These results stress the importance of Δ12 FAD in the regulation of cold adaptation processes, as well as the suitability of T. thermophila as a valuable model to investigate the regulation of membrane lipids and evolutionary conservation and divergence of the underlying mechanisms.     

Partial purification and characterization of β-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium,Pseudomonas 135

Summary An intracellular enzyme, d(—)--hydroxybutyric acid dehydrogenase involved in an intracellular poly-d(—)--hydroxybutyric acid degredation was isolated from a facultative methylotrophic bacterium, Pseudomonas 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to 11.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of d(—)--hydroxybutyric acid (D-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5–6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at — 20° C. The K _m values for oxidation and reduction reactions were determined as 1.84 mm for D-HB, 0.244 mm for NAD⁺, 0.319 mm for acetoacetate and 0.032 mm for NADH, respectively. It was also found that d-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mm for d-lactate, 0.196 mm for NADH and 1.82 mm for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive. Correspondence to: J. M. Lebeault     

Identification and characterization of Δ12 and Δ12/Δ15 bifunctional fatty acid desaturases in the oleaginous yeast Lipomyces starkeyi

Fatty acid desaturases play vital roles in the synthesis of unsaturated fatty acids. In this study, Δ12 and Δ12/Δ15 fatty acid desaturases of the oleaginous yeast Lipomyces starkeyi, termed LsFad2 and LsFad3, respectively, were identified and characterized. Saccharomyces cerevisiae expressing LsFAD2 converted oleic acid (C18:1) to linoleic acid (C18:2), while a strain of LsFAD3-expressing S. cerevisiae converted oleic acid to linoleic acid, and linoleic acid to α-linolenic acid (C18:3), indicating that LsFad2 and LsFad3 were Δ12 and bifunctional Δ12/Δ15 fatty acid desaturases, respectively. The overexpression of LsFAD2 in L. starkeyi caused an accumulation of linoleic acid and a reduction in oleic acid levels. In contrast, overexpression of LsFAD3 induced the production of α-linolenic acid. Deletion of LsFAD2 and LsFAD3 induced the accumulation of oleic acid and linoleic acid, respectively. Our findings are significant for the commercial production of polyunsaturated fatty acids, such as ω-3 polyunsaturated fatty acids, in L. starkeyi.

     

Functional screening of a novel Δ15 fatty acid desaturase from the coccolithophorid Emiliania huxleyi

The coccolithophorid Emiliania huxleyi is a bloom-forming marine phytoplankton thought to play a key role as a biological pump that transfers carbon from the surface to the bottom of the ocean, thus contributing to the global carbon cycle. This alga is also known to accumulate a variety of polyunsaturated fatty acids. At 25 °C, E. huxleyi produces mainly 14:0, 18:4n − 3, 18:5n − 3 and 22:6n − 3. When the cells were transferred from 25 °C to 15 °C, the amount of unsaturated fatty acids, i.e. 18:1n − 9, 18:3n − 3 and 18:5n − 3, gradually increased. Among the predicted desaturase genes whose expression levels were up-regulated at low temperature, we identified a gene encoding novel ?15 fatty acid desaturase, EhDES15, involved in the production of n − 3 polyunsaturated fatty acids in E. huxleyi. This desaturase contains a putative transit sequence for localization in chloroplasts and a ?6 desaturase-like domain, but it does not contain a cytochrome b₅ domain nor typical His-boxes found in ?15 desaturases. Heterologous expression of EhDES15 cDNA in cyanobacterium Synechocystis sp. PCC 6803 cells increased the level of n − 3 fatty acid species, which are produced at low levels in wild-type cells grown at 30 °C. The orthologous genes are only conserved in the genomes of prasinophytes and cryptophytes. The His-boxes conserved in orthologues varied from that of the canonical ?15 desaturases. These results suggested the gene encodes a novel ?15 desaturase responsible for the synthesis of 18:3n − 3 from 18:2n − 6 in E. huxleyi.     

Cold-induced accumulation of ω-3 polyunsaturated fatty acid in a liverwort, Marchantia polymorpha L

The liverwort Marchantia polymorpha L. synthesizes various long-chain polyunsaturated fatty acids including arachidonic acid and eicosapentaenoic acid, neither of which is produced by higher plants. Here we report the effects of temperature on long-chain polyunsaturated fatty acid accumulation in the liverwort. The accumulation of ω-3 polyunsaturated fatty acids increased significantly as the growth temperature decreased. Specifically, the relative content of eicosapentaenoic acid to total fatty acids at 5 °C was approximately 3-fold higher than at 25 °C. On the other hand, the accumulation of ω-6 polyunsaturated fatty acids decreased at low temperatures. An analysis of gene expression indicated that the mRNA of the MpFAD3 gene for ER ω-3 desaturase increased significantly at 5 °C. These results indicate that in the liverwort the n-3 pathway was enhanced at low temperature, mainly via expression of the cold-induced ω-3 desaturase gene, leading to increased accumulation of eicosapentaenoic acid.     

Free fatty acid receptor 4-β-arrestin 2 pathway mediates the effects of different classes of unsaturated fatty acids in osteoclasts and osteoblasts

Bone is a dynamic tissue that is constantly remodelled by bone resorbing osteoclasts and bone forming osteoblasts, respectively. A breakdown in the remodelling process underlies several bone diseases such as osteoporosis. Unsaturated fatty acids (UFAs) have been shown to have beneficial effects on bone health. However, the mechanism of action of UFAs in bone remains unclear. Free fatty acid receptor 4 (FFAR4) is expressed in bone cells and preferentially binds ω−3 and ω−7 UFAs. Therefore, we sought to determine if FFAR4 influenced the action of different classes of UFAs in bone cells. FFAR4 and potential signalling pathways, β-arrestin 2 (βarr2) and G_αq, were silenced in RAW264.7 murine macrophages (pre-osteoclasts) and MC3T3-E1 murine pre-osteoblasts. Cell differentiation, activation of signalling pathways and expression of regulatory genes were evaluated. The ω−3 UFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and the ω−7 UFA, palmitoleic acid (PLA), were shown to require the FFAR4/βarr2 signalling pathway to inhibit osteoclast differentiation in RAW264.7 murine macrophages. The ω−6 UFA, arachidonic acid, and the ω−9 UFA, oleic acid (OA), were shown to inhibit osteoclast formation but did not use FFAR4. DHA, EPA, PLA and OA enhanced osteoblast signalling through the FFAR4/βarr2 signalling axis. This study reveals that FFAR4/βarr2 signalling may mediate the bone protective effects of different classes of UFAs in osteoclasts and osteoblasts.     

Higher plasma n-3 fatty acid status in the moderately healthy elderly in southern Québec: Higher fish intake or aging-related change in n-3 fatty acid metabolism?

The elderly reportedly have a significantly higher % of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids in plasma and red cell lipids. However, these observations are from a few small studies and the health status of the elderly in these studies is for the most part unclear. Since the elderly are susceptible to cardiovascular and neurological illnesses that seem to be related in part to lower intake of n-3 fatty acids it seems paradoxical that their blood levels of EPA and DHA would be higher than in young adults. We report here plasma fatty acid profiles and their response to supplementation with two types of fish oils from several of our recent studies in the moderately healthy elderly. We define the moderately healthy elderly as those who were in good physical condition, had no cognitive decline and, if present, in whom hypothyroidism, hyperlipidemia and/or hypertension were well-controlled. As shown previously, we confirm the higher % EPA and % total n-3 fatty acids (but not DHA) in fasting plasma and extend these findings to include higher plasma concentrations (mg/L) of n-3 fatty acids as well. The EPA-predominant supplement raised DHA only in the young, whereas the DHA-predominant supplement raised EPA more in the young than in the elderly. The moderately healthy elderly clearly have higher plasma n-3 fatty acids but whether this reflects differences in intake versus aging-related changes in n-3 fatty acid metabolism remains to be elucidated.     

Purification and characterization of a novel l-2-amino-Δ2-thiazoline-4-carboxylic acid hydrolase from Pseudomonas sp. strain ON-4a expressed in E. coli

l-2-Amino-Δ²-thiazoline-4-carboxylic acid hydrolase (ATC hydrolase) was purified and characterized from the crude extract of Escherichia coli, in which the gene for ATC hydrolase of Pseudomonas sp. strain ON-4a was expressed. The results of SDS–polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200 suggested that the ATC hydrolase was a tetrameric enzyme consisted of identical 25-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 7.0 and 30–35°C, respectively. The enzyme did not require divalent cations for the expression of the activity, and Cu²⁺ and Mn²⁺ ions strongly inhibited the enzyme activity. An inhibition experiment by diethylpyrocarbonic acid, 2-hydroxy-5-nitrobenzyl bromide, and N-bromosuccinimide suggested that tryptophan, cysteine, or/and histidine residues may be involved in the catalytic site of this enzyme. The enzyme was strictly specific for the l-form of d,l-ATC and exhibited high activity for the hydrolysis of l-ATC with the values of K _m (0.35 mM) and V _max (69.0 U/mg protein). This enzyme could not cleave the ring structure of derivatives of thiazole, thiazoline, and thiazolidine tested, except for d,l- and l-ATC. These results show that the ATC hydrolase is a novel enzyme cleaving the carbon–sulfur bond in a ring structure of l-ATC to produce N-carbamoyl-l-cysteine.