Cloning and expression of the multifunctional human fatty acid synthase and its subdomains in Escherichia coli

We engineered a full-length (8.3-kbp) cDNA coding for fatty acid synthase (FAS; EC 2.3.1.85) from the human brain FAS cDNA clones we characterized previously. In the process of accomplishing this task, we developed a novel PCR procedure, recombinant PCR, which is very useful in joining two overlapping DNA fragments that do not have a common or unique restriction site. The full-length cDNA was cloned in pMAL-c2 for heterologous expression in Escherichia coli as a maltose-binding protein fusion. The recombinant protein was purified by using amylose-resin affinity and hydroxylapatite chromatography. As expected from the coding capacity of the cDNA expressed, the chimeric recombinant protein has a molecular weight of 310,000 and reacts with antibodies against both human FAS and maltose-binding protein. The maltose-binding protein-human FAS (MBP-hFAS) catalyzed palmitate synthesis from acetyl-CoA, malonyl-CoA, and NADPH and exhibited all of the partial activities of FAS at levels comparable with those of the native human enzyme purified from HepG2 cells. Like the native HepG2 FAS, the products of MBP-hFAS are mainly palmitic acid (>90%) and minimal amounts of stearic and arachidic acids. Similarly, a human FAS cDNA encoding domain I (β-ketoacyl synthase, acetyl-CoA and malonyl-CoA transacylases, and β-hydroxyacyl dehydratase) was cloned and expressed in E. coli using pMAL-c2. The expressed fusion protein, MBP-hFAS domain I, was purified to apparent homogeneity (Mr 190,000) and exhibited the activities of the acetyl/malonyl transacylases and the β-hydroxyacyl dehydratase. In addition, a human FAS cDNA encoding domains II and III (enoyl and β-ketoacyl reductases, acyl carrier protein, and thioesterase) was cloned in pET-32b(+) and expressed in E. coli as a fusion protein with thioredoxin and six in-frame histidine residues. The recombinant fusion protein, thioredoxin-human FAS domains II and III, that was purified from E. coli had a molecular weight of 159,000 and exhibited the activities of the enoyl and β-ketoacyl reductases and the thioesterase. Both the MBP and the thioredoxin-His-tags do not appear to interfere with the catalytic activity of human FAS or its partial activities.

Aspergillus has distinct fatty acid synthases for primary and secondary metabolism

Aspergillus nidulans contains two functionally distinct fatty acid synthases (FASs): one required for primary fatty acid metabolism (FAS) and the other required for secondary metabolism (sFAS). FAS mutants require long-chain fatty acids for growth, whereas sFAS mutants grow normally but cannot synthesize sterigmatocystin (ST), a carcinogenic secondary metabolite structurally and biosynthetically related to aflatoxin. sFAS mutants regain the ability to synthesize ST when provided with hexanoic acid, supporting the model that the ST polyketide synthase uses this short-chain fatty acid as a starter unit. The characterization of both the polyketide synthase and FAS may provide novel means for modifying secondary metabolites.

Nutritional regulation of the fatty acid synthase promoter in vivo: Sterol regulatory element binding protein functions through an upstream region containing a sterol regulatory element

The transcription of fatty acid synthase (FAS), a central enzyme in de novo lipogenesis, is dramatically induced by fasting/refeeding and insulin. We reported that upstream stimulatory factor binding to the −65 E-box is required for induction of the FAS transcription by insulin in 3T3-L1 adipocytes. On the other hand, we recently found that two upstream 5′ regions are required for induction in vivo by fasting/refeeding and insulin; one at −278 to −131 albeit at a low level, and the other at −444 to −278 with an E-box at −332 where upstream stimulatory factor functions for maximal induction. Here, we generated double transgenic mice carrying the chloramphenicol acetyltransferase reporter driven by the various 5′ deletions of the FAS promoter region and a truncated active form of the sterol regulatory element (SRE) binding protein (SREBP)-1a. We found that SREBP participates in the nutritional regulation of the FAS promoter and that the region between −278 and −131 bp is required for SREBP function. We demonstrate that SREBP binds the −150 canonical SRE present between −278 and −131, and SREBP can function through the −150 SRE in cultured cells. These in vivo and in vitro results indicate that SREBP is involved in the nutritional induction of the FAS promoter via the −278/−131 region and that the −150 SRE is the target sequence.

Targeted disruption of mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation

Abnormalities of fatty acid metabolism are recognized to play a significant role in human disease, but the mechanisms remain poorly understood. Long-chain acyl-CoA dehydrogenase (LCAD) catalyzes the initial step in mitochondrial fatty acid oxidation (FAO). We produced a mouse model of LCAD deficiency with severely impaired FAO. Matings between LCAD +/− mice yielded an abnormally low number of LCAD +/− and −/− offspring, indicating frequent gestational loss. LCAD −/− mice that reached birth appeared normal, but had severely reduced fasting tolerance with hepatic and cardiac lipidosis, hypoglycemia, elevated serum free fatty acids, and nonketotic dicarboxylic aciduria. Approximately 10% of adult LCAD −/− males developed cardiomyopathy, and sudden death was observed in 4 of 75 LCAD −/− mice. These results demonstrate the crucial roles of mitochondrial FAO and LCAD in vivo.

A 20-nucleotide element in the intestinal fatty acid binding protein gene modulates its cell lineage-specific, differentiation-dependent, and cephalocaudal patterns of expression in transgenic mice.

A sequence of epithelial cell proliferation, allocation to four principal lineages, migration-associated differentiation, and cell loss occurs along the crypt-villus axis of the mouse intestine. The sequence is completed in a few days and is recapitulated throughout the life-span of the animal. We have used an intestine-specific fatty acid binding protein gene, Fabpi, as a model for studying regulation of gene expression in this unique developmental system. Promoter mapping studies in transgenic mice identified a 20-bp cis-acting element (5'-AGGTGGAAGCCATCACACTT-3') that binds small intestinal nuclear proteins and participates in the control of Fabpi's cephalocaudal, differentiation-dependent, and cell lineage-specific patterns of expression. Immunocytochemical studies using confocal and electron microscopy indicate that it does so by acting as a suppressor of gene expression in the distal small intestine/colon, as a suppressor of gene activation in proliferating and nonproliferating cells located in the crypts of Lieberkühn, and as a suppressor of expression in the growth factor and defensin-producing Paneth cell lineage. The 20-bp domain has no obvious sequence similarities to known transcription factor binding sites. The three functions modulated by this compact element represent the types of functions required to establish and maintain the intestine's remarkably complex spatial patterns of gene expression. The transgenes described in this report also appear to be useful in characterizing the crypt's stem cell hierarchy.

Diatom abundance and fatty acid composition of ice algae and of Calanus glacialis in samples obtained in 2008 in the eastern Beaufort Sea

The copepod Calanus glacialis plays a key role in the lipid-based energy flux in Arctic shelf seas. By utilizing both ice algae and phytoplankton, this species is able to extend its growth season considerably in these seasonally ice-covered seas. This study investigated the impacts of the variability in timing and extent of the ice algal bloom on the reproduction and population success of C. glacialis. The vertical distribution, reproduction, amount of storage lipids, stable isotopes, fatty acid and fatty alcohol composition of C. glacialis were assessed during the Circumpolar Flaw Lead System Study. Data were collected in the Amundsen Gulf, south-eastern Beaufort Sea, from January to July 2008 with the core-sampling from March to April. The reduction in sea ice thickness and coverage observed in the Amundsen Gulf in 2007 and 2008 affected the life strategy and reproduction of C. glacialis. Developmental stages CIII and CIV dominated the overwintering population, which resulted in the presence of very few CV and females during spring 2008. Spawning began at the peak of the ice algal bloom that preceded the precocious May ice break-up. Although the main recruitment may have occurred later in the season, low abundance of females combined with a potential mismatch between egg production/development to the first feeding stage and phytoplankton bloom resulted in low recruitment of C. glacialis in the early summer of 2008.

Effect of a protein and energy dense n-3 fatty acid enriched oral supplement on loss of weight and lean tissue in cancer cachexia:A randomised double blind trial

Aim: N-3 fatty acids, especially eicosapentaenoic acid (EPA), may possess anticachectic properties. This trial compared a protein and energy dense supplement enriched with n-3 fatty acids and antioxidants (experimental: E) with an isocaloric isonitrogenous control supplement (C) for their effects on weight, lean body mass (LBM), dietary intake, and quality of life in cachectic patients with advanced pancreatic cancer. Methods: A total of 200 patients (95 E; 105 C) were randomised to consume two cans/day of the E or C supplement (480 ml, 620 kcal, 32 g protein ± 2.2 g EPA) for eight weeks in a multicentre, randomised, double blind trial. Results: At enrolment, patients' mean rate of weight loss was 3.3 kg/month. Intake of the supplements (E or C) was below the recommended dose (2 cans/day) and averaged 1.4 cans/day. Over eight weeks, patients in both groups stopped losing weight (Δweight E: -0.25 kg/month versus C: -0.37 kg/month; p=0.74) and LBM (ΔLBM E: +0.27 kg/month versus C: +0.12 kg/month; p=0.88) to an equal degree (change from baseline E and C, p<0.001). In view of evident non-compliance in both E and C groups, correlation analyses were undertaken to examine for potential dose-response relationships. E patients demonstrated significant correlations between their supplement intake and weight gain (r=0.50, p<0.001) and increase in LBM (r=0.33, p=0.036). Such correlations were not statistically significant in C patients. The relationship of supplement intake with change in LBM was significantly different between E and C patients (p=0.043). Increased plasma EPA levels in the E group were associated with weight and LBM gain (r=0.50, p<0.001; r=0.51, p=0.001). Weight gain was associated with improved quality of life (p<0.01) only in the E group. Conclusion: Intention to treat group comparisons indicated that at the mean dose taken, enrichment with n-3 fatty acids did not provide a therapeutic advantage and that both supplements were equally effective in arresting weight loss. Post hoc dose-response analysis suggests that if taken in sufficient quantity, only the n-3 fatty acid enriched energy and protein dense supplement results in net gain of weight, lean tissue, and improved quality of life. Further trials are required to examine the potential role of n-3 enriched supplements in the treatment of cancer cachexia.

Polyunsaturated fatty acid requirements of murine colon adenocarcinomas

The polyunsaturated fatty acid (PUFA) requirements of three transplantable murine colon adenocarcinomas, the MAC13, MAC16 and MAC26, were evaluated in vitro and in vivo. When serum concentrations became growth limiting in vitro, proliferation of the MAC13 and MAC26 cell lines was stimulated by linoleic acid (LA) at 18μM and arachidonic acid (AA) at 16 or 33μM respectively. This was not demonstrated by the MAC16 cell line. MAC13 and MAC26 cells were found to be biochemically fatty acid deficient as measured by the formation of Mead acid (20:3 n-9), but the MAC16 cells were not. In vivo the growth of the MAC26 tumour was stimulated by daily oral administration of LA between 0.4-2.0g/kg. There was a threshold value of 0.4g/kg for the stimulation of MAC26 tumour growth, above which there was no further increase in tumour growth, and below which no increase in tumour growth was observed. This increased tumour growth was due to the stimulation of tumour cell proliferation in all areas of the tumour, with no effect on the cell loss factor. The growth of the MAC13, MAC16, and MAC26 cell lines in vitro were more effectively inhibited by lipoxygenase (LO) inhibitors than the cyclooxygenase inhibitor indomethacin. The specific 5-LO inhibitor Zileuton and the leukotriene D4 antagonist L-660,711 were less effective inhibitors of MAC cell growth in vitro than the less specific LO inhibitors BWA4C, BWB70C and CV6504. Studies of the hyroxyeicosatetraenoic acids (HETEs) produced from exogenous AA in these cells, suggested that a balance of eicosanoids produced from 5-LO, 12-LO and 15-LO pathways was required for cell proliferation. In vivo BWA4C, BWB70C and CV6504 demonstrated antitumour action against the MAC26 tumour between 20-50mg/kg/day. CV6504 also inhibited the growth of the MAC 13 tumour in vivo with an optimal effect between 5-10mg/kg/day. The antitumour action against the MAC16 tumour was also accompanied by a reduction in the tumour-induced host body weight loss at 10-25mg/kg/day. The antitumour action of CV6504 in all three tumour models was partially reversed by daily oral administration of 1.0g/kg LA. Studies of the AA metabolism in tumour homogenates suggested that this profound antitumour action, against what are generally chemoresistant tumours, was due to inhibition of eicosanoid production through LO pathways. As a result of these studies, CV6504 has been proposed for stage I./II. clinical trials against pancreatic cancer by the Cancer Research Campaign. This will be the first LO inhibitor entering the clinic as a therapeutic agent.

Effect of cancer cachexia on triacylglycerol/fatty acid substrate cycling in white adipose tissue

The effect of cancer cachexia on the TAG/FA substrate cycle in white adipose tissue was determined in vivo using the MAC16 murine model of cachexia. When compared with non-tumor-bearing animals, the rate of TAG-glycerol production was found to be increased almost threefold in animals bearing the MAC13 tumor, which does not induce cachexia, but was not further elevated in animals bearing the MAC16 tumor. In both cases TAG-glycerol production and de novo synthesis of TAG-FA were also increased above non-tumor-bearing animals. In animals bearing the MAC16 tumor, the TAG-FA rates were significantly higher than in animals bearing the MAC13 tumor. This suggests that the presence of the tumor alone is sufficient to cause an increase in cycling rate, and in the absence of an elevated energy intake (MAC16) this may contribute to the depletion of adipose tissue.

Total phospholipid fatty acid concentrations structrures of ODP Hole 191-1179 samples (Table 1)

Sediment samples ranging from 0.05 to 278 m below sea floor (mbsf) at a Northwest Pacific deep-water (5564 mbsl) site (ODP Leg 191, Site 1179) were analyzed for phospholipid fatty acids (PLFAs). Total PLFA concentrations decreased by a factor of three over the first meter of sediment and then decreased at a slower rate to approximately 30 mbsf. The sharp decrease over the first meter corresponds to the depth of nitrate and Mn(IV) reduction as indicated by pore water chemistry. PLFA-based cell numbers at site 1179 had a similar depth profile as that for Acridine orange direct cell counts previously made on ODP site 1149 sediments which have a similar water depth and lithology. The mole percentage of straight chain saturated PLFAs increases with depth, with a large shift between the 0.95 and 3.95 mbsf samples. PLFA stable carbon isotope ratios were determined for sediments from 0.05 to 4.53 mbsf and showed a general trend toward more depleted d13C values with depth. Both of these observations may indicate a shift in the bacterial community with depth across the different redox zones inferred from pore water chemistry data. The PLFA 10me16:0, which has been attributed to the bacterial genera Desulfobacter in many marine sediments, showed the greatest isotopic depletion, decreasing from -20 to -35 per mil over the first meter of sediment. Pore water chemistry suggested that sulfate reduction was absent or minimal over this same sediment interval. However, 10me16:0 has been shown to be produced by recently discovered anaerobic ammonium oxidizing (anammox) bacteria which are known chemoautotrophs. The increasing depletion in d13C of 10me16:0 with the unusually lower concentration of ammonium and linear decrease of nitrate concentration is consistent with a scenario of anammox bacteria mediating the oxidation of ammonium via nitrite, an intermediate of nitrate reduction.

Gut content, fatty acid composition and metabolic rates of the amphipod Anonyx sarsi during POLARSTERN cruise ARK-XIX/1, ice station PS64/070-6

During a winter expedition to the western Barents Sea in March 2003, benthic amphipods of the species Anonyx sarsi were observed directly below pack ice. Only males and juveniles [16.0-37.0 mm long, 16.2-120.8 mg dry mass (DM)] were collected. Guts contained macroalgal fibres, fish eggs and flesh from large carrion. Amphipods had very low levels of total lipids (2.7-17.2% DM). Analysis of lipid biomarkers showed that some of the specimens had preyed on pelagic copepods. Individual respiration rates ranged over 0.4-1.7 ml O2/day (mean: 1.2 ml, SD: 0.5 ml). Individual ammonia excretion rates varied between 7.8 µg and 49.3 µg N/day (mean: 30.7 µg, SD: 15.2 µg). The atomic O:N ratio ranged over 35 to 71 (mean: 55, SD: 14), indicating lipid-dominated metabolism. Mass-specific respiration ranged over 9.8-16.6 ml O2/day/g DM (mean: 13.1 ml, SD: 2.2 ml). The metabolic rates of A. sarsi were twice as high as those of the truly sympagic amphipod Gammarus wilkitzkii, which is better adapted to the under-ice habitat by its energy-saving attached lifestyle. It is concluded that males and juveniles of A. sarsi were actively searching for food in the water column and at the ice underside, but that the nutritional status of the amphipods in late Arctic winter was generally very poor.

Geochemistry, fatty acid content and phylogenetic affiliation of soil samples from Livingston Island, South Shetland Archipelago, Antarctica

Microorganisms inhabit very different soil habitats in the ice-free areas of Antarctica, playing a major role in nutrient cycling in cold environments. We studied the soil characteristics and the dominant bacterial composition from nine different soil profiles located on Livingston Island (maritime Antarctica). The total carbon (TC) and total nitrogen (TN) values were high for the vegetated soils, decreasing with depth, whereas the values for the mineral soils were generally low. Soil pH was more acidic for moss-covered soils and neutral to alkaline for mineral soils. Numbers of culturable heterotrophic bacteria were higher at vegetated sites, but significant numbers were also detectable in carbon-depleted soils. Patterns of denaturing gradient gel electrophoresis (DGGE) revealed a highly heterogeneous picture throughout the soil profiles. Subsequent sequencing of DGGE bands revealed in total 252 sequences that could be assigned to 114 operational taxonomic units, showing the dominance of members of the Bacteroidetes and Acidobacteria. The results of phospholipid fatty acid analysis showed a lack of unsaturated fatty acids for most of the samples. Samples with a prevalence of unsaturated over saturated fatty acids were restricted to several surface samples. Statistical analysis showed that the dominant soil bacterial community composition is most affected by TC and TN contents and soil physical factors such as grain size and moisture, but not pH. Keywords

(Table 2) Fatty acid composition of polychaetes sampled during POLARSTERN cruise ANT-XXIV/2

The fatty acid (FA) composition of representatives belonging to 18 polychaete families from the Southern Ocean shelf and deep sea (600 to 5337 m) was analysed in order to identify trophic biomarkers and elucidate possible feeding preferences. Total FA content was relatively low with few exceptions and ranged from 1.0 to 11.6% of total body dry weight. The most prominent FA found were 20:5(n-3), 16:0, 22:6(n-3), 18:1(n-7), 20:4(n-6), 18:0, 20:1(n-11) and 18:1(n-9). For some polychaete families and species FA profiles indicated selective feeding on certain dietary components, like freshly deposited diatom remains (e.g., Spionidae, Fauveliopsidae and Flabelligeridae) or foraminiferans (e.g., Euphrosinidae, Nephtyidae and Syllidae). Feeding patterns were relatively consistent within families at the deep stations, while the FA composition differed between the deep and the shelf stations within the same family. Fatty alcohols, indicative of wax ester storage, were found in almost all families (in proportions of 0.0 to 29.3% of total FA and fatty alcohols). The development of this long-term storage mechanism of energy reserves possibly displays an evolutionary strategy.

Phospholipid fatty acid and quinone composition of sediments from ODP Leg 205 sites

Assessing the habitability of deep-sea sediments undergoing compaction, compression, and subduction at convergent margins adds to our understanding of the limits of the terrestrial biosphere. In this work, we report exploratory biomarker data on sediments obtained at Ocean Drilling Program (ODP) Sites 1253, 1254, and 1255 during drilling at the Costa Rica subduction trench and forearc sedimentary wedge. The samples selected for postcruise biomarker analyses were located within intervals of potentially enhanced fluid flow within the décollement and sedimentary wedge fault zones (Sites 1254 and 1255) and within basal carbonates at the reference site (Site 1253). The passage of fluids that are geochemically distinct from ambient interstitial water provides a disequilibrium setting that may enhance habitability. Biomarker data show low levels of microbial biomass in subseafloor sediments sampled at the Costa Rica convergent margin as deep as ~370 meters below seafloor.

Fatty acid composition of tissue samples from Arctic walruses (Odobenus rosmarus rosmarus) and their prey

Blubber biopsies were collected from 18 adult male walruses from Svalbard, Norway. The biopsies were taken vertically through the skin and the entire blubber layer down to, but not including, the muscle layer. Fatty acid (FA) compositions of inner blubber, outer blubber and dermis of the walruses and of potential prey organisms were determined. The three layers differed significantly from one another in FA composition. Generally, the inner blubber contained more long-chained monounsaturated, saturated and polyunsaturated FAs, while the outer blubber and dermis contained more short-chained monounsaturated FAs. This stratification is similar to what has been observed in other marine mammal species. However, differences between layers were less pronounced than in most other species, possibly because the extremely thick overlying dermis of walruses provides an insulating shield, which affects the FA composition of the outer blubber. The FA composition of the potential prey organisms was different from that of the blubber of the walruses, although more similar to the inner blubber than to the outer blubber or dermis. FA composition in the inner blubber was not significantly correlated with age (assessed by tusk volume), while the FA composition of the outer blubber and dermis were significantly correlated with age.