Rainbow trout (Oncorhynchus mykiss) Elovl5 and Elovl2 differ in selectivity for elongation of omega-3 docosapentaenoic acid

The synthesis of the omega-3 long-chain polyunsaturated fatty acids (LCPUFA)  eicosapentaenoic acid (EPA; 20:5n− 3) and docosahexaenoic acid (DHA; 22:6n − 3) from dietary α-linolenic acid (ALA; 18:3n − 3) requires three desaturation and three elongation steps in vertebrates. The elongation of EPA to docosapentaenoic acid (DPA; 22:5n − 3) can be catalysed by the elongase enzymes Elovl5 or Elovl2, but further elongation of DPA to 24:5n − 3, the penultimate precursor of DHA, is limited to Elovl2, at least in mammals. Elovl5 enzymes have been characterised from seventeen fish species but Elovl2 enzymes have only been characterised in two of these fish. The essentiality of Elovl2 for DHA synthesis is unknown in fish. This study is the first to identify an Elovl2 in rainbow trout (Oncorhynchus mykiss) and functionally characterise the Elovl5 and Elovl2 using a yeast expression system. Elovl5 was active with C_18–20 PUFA substrates and not C₂₂ PUFA. In contrast, Elovl2 was active with C_20–22 PUFA substrates and not C₁₈ PUFA. Thus, rainbow trout is dependent on Elovl2 for DPA to 24:5n − 3 synthesis and ultimately DHA synthesis. The expression of elovl5 was significantly higher than elovl2 in liver. Elucidating this dependence on Elovl2 to elongate DPA and the low elovl2 gene expression compared with elovl5 are critical findings in understanding the potential for rainbow trout to synthesize DHA.     

N − 3PUFA differentially modulate palmitate-induced lipotoxicity through alterations of its metabolism in C2C12 muscle cells

Excessive energy intake leads to fat overload and the formation of lipotoxic compounds mainly derived from the saturated fatty acid palmitate (PAL), thus promoting insulin resistance (IR) in skeletal muscle. N − 3 polyunsaturated fatty acids (n − 3PUFA) may prevent lipotoxicity and IR. The purpose of this study was to examine the differential effects of n − 3PUFA on fatty acid metabolism and insulin sensitivity in muscle cells. C2C12 myotubes were treated with 500 μM of PAL without or with 50 μM of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) for 16 h. PAL decreased insulin-dependent AKT activation and glucose uptake and increased the synthesis of ceramides and diglycerides (DG) derivatives, leading to protein kinase Cθ activation. EPA and DHA, but not ALA, prevented PAL-decreased AKT activation but glucose uptake was restored to control values by all n − 3PUFA vs. PAL. Total DG and ceramide contents were decreased by all n − 3PUFA, but only EPA and DHA increased PAL β-oxidation, decreased PAL incorporation into DG and reduced protein kinase Cθ activation. EPA and DHA emerge as better candidates than ALA to improve fatty acid metabolism in skeletal muscle cells, notably via their ability to increase mitochondrial β-oxidation.     

Mammalian ALOX15 orthologs exhibit pronounced dual positional specificity with docosahexaenoic acid

Mammalian lipoxygenases (LOX) have been implicated in cell differentiation and in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. Although the reaction specificity of mammalian LOX with n − 6 fatty acids (linoleic acid, arachidonic acid) has been explored in detail little information is currently available on the product patterns formed from n − 3 polyenoic fatty acids, which are of particular nutritional importance and serve as substrate for the biosynthesis of pro-resolving inflammatory mediators such as resolvins and maresins. Here we expressed the ALOX15 orthologs of eight different mammalian species as well as human ALOX12 and ALOX15B as recombinant his-tag fusion proteins and characterized their reaction specificity with the most abundantly occurring polyunsaturated fatty acids (PUFAs) including 5,8,11,14,17-eicosapentaenoic acid (EPA) and 4,7,10,13,16,19-docosahexaenoic acid (DHA). We found that the LOX isoforms tested accept these fatty acids as suitable substrates and oxygenate them with variable positional specificity to the corresponding n − 6 and n − 9 hydroperoxy derivatives. Surprisingly, human ALOX15 as well as the corresponding orthologs of chimpanzee and orangutan, which oxygenates arachidonic acid mainly to 15S-H(p)ETE, exhibit a pronounced dual reaction specificity with DHA forming similar amounts of 14- and 17-H(p)DHA. Moreover, ALOX15 orthologs prefer DHA and EPA over AA when equimolar concentrations of n − 3 and n − 6 PUFA were supplied simultaneously. Taken together, these data indicate that the reaction specificity of mammalian LOX isoforms is variable and strongly depends on the chemistry of fatty acid substrates. Most mammalian ALOX15 orthologs exhibit dual positional specificity with highly unsaturated n − 3 polyunsaturated fatty acids.     

Assessment of a land-locked Atlantic salmon (Salmo salar L.) population as a potential genetic resource with a focus on long-chain polyunsaturated fatty acid biosynthesis

The natural food for Atlantic salmon (Salmo salar) in freshwater has relatively lower levels of omega − 3 (n − 3) long-chain polyunsaturated fatty acids (LC-PUFA) than found in prey for post-smolt salmon in seawater. Land-locked salmon such as the Gullspång population feed exclusively on freshwater type lipids during its entire life cycle, a successful adaptation derived from divergent evolution. Studying land-locked populations may provide insights into the molecular and genetic control mechanisms that determine and regulate n − 3 LC-PUFA biosynthesis and retention in Atlantic salmon. A two factorial study was performed comparing land-locked and farmed salmon parr fed diets formulated with fish or rapeseed oil for 8 weeks. The land-locked parr had higher capacity to synthesise n − 3 LC-PUFA as indicated by higher expression and activity of desaturase and elongase enzymes. The data suggested that the land-locked salmon had reduced sensitivity to dietary fatty acid composition and that dietary docosahexaenoic acid (DHA) did not appear to suppress expression of LC-PUFA biosynthetic genes or activity of the biosynthesis pathway, probably an evolutionary adaptation to a natural diet lower in DHA. Increased biosynthetic activity did not translate to enhanced n − 3 LC-PUFA contents in the flesh and diet was the only factor affecting this parameter. Additionally, high lipogenic and glycolytic potentials were found in land-locked salmon, together with decreased lipolysis which in turn could indicate increased use of carbohydrates as an energy source and a sparing of lipid.     

Fatty acid profiles,growth, and immune responses of neonatal lambs fed milk replacer and supplemented with fish oil or safflower oil

Diets supplemented with long chain, n − 3 polyunsaturated fatty acids (PUFA) have improved the health and performance of neonatal and growing animals. This study was conducted with lambs that were orphaned at approximately 1 day of age to determine whether supplementing milk replacer fed lambs with oils rich in long chain n − 3 or n − 6 PUFA would alter plasma lipid profiles and affect growth characteristics and immune functions. From days 1 to 28 of age, lambs had ad libitum access to commercial milk replacer. From days 7 to 28 of age, lambs received twice daily either 1 g of soybean oil, 1 g of fish oil, or 1 g of safflower oil per os in a gelatin capsule (n = 60 pens; 20 pens/treatment; one ewe and one ram with similar initial body weights/pen). On days 7, 14, 21, and 28 of age, lambs were weighed, and jugular blood was collected from ram lambs. Lymphocyte proliferation in vitro, differential white blood cell (WBC) counts, and weight gains were quantified. Plasma from days 7 and 28 was used for fatty acid analyses. Fish oil increased (P < 0.001) plasma total n − 3 fatty acid concentration and total n − 3:total n − 6 fatty acid ratio. Pen body weight (i.e., total lamb weight per pen) increased (P < 0.001) with day (day 7, 11.9 kg; day 14, 15.1 kg; day 21, 18.2 kg; and day 28, 21.2 kg), but oil treatment did not affect pen body weight. Neither oil treatment, day, nor oil treatment × day interaction were significant for pen body weight gains (3.5 kg), pen average daily gains (0.5 kg), pen milk intakes (19.0 kg), or pen gain:feed ratio (0.18) measured during three intervals: days 7–14; days 14–21; and days 21–28. Day, but not oil treatment, affected (P < 0.001) unstimulated, concanavalin A stimulated, and lipopolysaccharides stimulated lymphocyte proliferation: days 14, 21, and 28 proliferation > day 7 proliferation. For neutrophils per 100 WBC, the treatment × day interaction was significant (P < 0.05). Oil treatment and day affected (P < 0.01 and <0.05, respectively) lymphocyte numbers per 100 WBC. For monocytes, eosinophils, and basophils, neither oil treatment, day, nor the oil treatment × day interaction were significant. Fish oil altered plasma fatty acid profiles, but it did not seem to improve measures of the performance or immune function of healthy, milk replacer fed lambs.     

Suppression of VLDL secretion by cultured hepatocytes incubated with chylomicron remnants enriched in n−3 polyunsaturated fatty acids is regulated by hepatic nuclear factor-4α

Dietary n? 3 polyunsaturated fatty acids (PUFA) suppress the secretion of very low density lipoprotein (VLDL) directly when delivered to the liver in chylomicron remnants (CMR). The role of sterol regulatory element-binding proteins (SREBPs) and hepatic nuclear factor-4α (HNF-4α) in the regulation of this effect was investigated. Chylomicron remnant-like particles (CRLPs) containing triacylglycerol (TG) from palm (rich in saturated fatty acids (SFA)) or fish (rich in n? 3 PUFA) oil were incubated with cultured rat hepatocytes (24 h) and the expression of protein and mRNA for SREBP-1, SREBP-2 and HNF-4α, and levels of mRNA for their target genes were determined. SREBP-1 and -2 protein expression in the membrane and nuclear fractions was unaffected by either type of CRLPs. mRNA abundance for SREBP-1c and -2 was also unchanged by CRLP-treatment, as were levels of mRNA for target genes of SREBP-1, including steroyl CoA desaturase, acetyl CoA carboxylase, fatty acid synthase and ATP citrate lyase, and SREBP-2 (3-hydroxy-3-methylglutaryl CoA reductase). In contrast, HNF-4α protein and mRNA levels were significantly decreased by CRLPs enriched in n? 3 PUFA, but not SFA, and the expression of mRNA for HNF-4α target genes, including HNF-1α, apolipoprotein B and the microsomal TG transfer protein, was also lowered by n? 3 PUFA-, but not SFA-enriched CRLPs. These findings suggest that the direct suppression of VLDL secretion by dietary n? 3 PUFA delivered to the liver in CMR is mediated via decreased expression of HNF-4α.     

Muscle fiber conduction velocity in different gait phases of early and late-stage diabetic neuropathy

We investigated the muscle fiber conduction velocity (MFCV) during gait phases of the lower limb muscles in individuals with various degrees of diabetic peripheral neuropathy (DPN). Forty-five patients were classified into severity degrees of DPN by a fuzzy model. The stages were absent (n = 11), mild (n = 14), moderate (n = 11) and severe (n = 9), with 10 matched healthy controls. While walking, all subjects had their sEMG (4 linear electrode arrays) recorded for tibialis anterior (TA), gastrocnemius medialis (GM), vastus lateralis (VL) and biceps femoris (BF). MFCV was calculated using a maximum likelihood algorithm with 30 ms standard deviation Gaussian windows. In general, individuals in the earlier stages of DPN showed lower MFCV of TA, GM and BF, whilst individuals with severe DPN presented higher MFCV of the same muscles. We observed that mild patients already showed lower MFCV of TA at early stance and swing, and lower MFCV of BF at swing. All diabetic groups showed a markedly reduction in MFCV of VL, irrespective of DPN. Severe patients presented higher MFCV mainly in distal muscles, TA at early and swing phases and GM at propulsion and midstance. The absent group already showed MFCV of VL and GM reductions at the propulsion phase and of VL at early stance. Although MFCV changes were not as progressive as the DPN was, we clearly distinguished diabetic patients from controls, and severe patients from all others.     

Matrix density alters zyxin phosphorylation,which limits peripheral process formation and extension in endothelial cells invading 3D collagen matrices

This study was designed to determine the optimal conditions required for known pro-angiogenic stimuli to elicit successful endothelial sprouting responses. We used an established, quantifiable model of endothelial cell (EC) sprout initiation where ECs were tested for invasion in low (1 mg/mL) and high density (5 mg/mL) 3D collagen matrices. Sphingosine 1-phosphate (S1P) alone, or S1P combined with stromal derived factor-1α (SDF) and phorbol ester (TPA), elicited robust sprouting responses. The ability of these factors to stimulate sprouting was more effective in higher density collagen matrices. S1P stimulation resulted in a significant increase in invasion distance, and with the exception of treatment groups containing phorbol ester, invasion distance was longer in 1 mg/mL compared to 5 mg/mL collagen matrices. Closer examination of cell morphology revealed that increasing matrix density and supplementing with SDF and TPA enhanced the formation of multicellular structures more closely resembling capillaries. TPA enhanced the frequency and size of lumen formation and correlated with a robust increase in phosphorylation of p42/p44 Erk kinase, while S1P and SDF did not. Also, a higher number of significantly longer extended processes formed in 5 mg/mL compared to 1 mg/mL collagen matrices. Because collagen matrices at higher density have been reported to be stiffer, we tested for changes in the mechanosensitive protein, zyxin. Interestingly, zyxin phosphorylation levels inversely correlated with matrix density, while levels of total zyxin did not change significantly. Immunofluorescence and localization studies revealed that total zyxin was distributed evenly throughout invading structures, while phosphorylated zyxin was slightly more intense in extended peripheral processes. Silencing zyxin expression increased extended process length and number of processes, while increasing zyxin levels decreased extended process length. Altogether these data indicate that ECs integrate signals from multiple exogenous factors, including changes in matrix density, to accomplish successful sprouting responses. We show here for the first time that zyxin limited the formation and extension of fine peripheral processes used by ECs for matrix interrogation, providing a molecular explanation for altered EC responses to high and low density collagen matrices.     

Novel insights into cyclooxygenases,linoleate diol synthases,and lipoxygenases from deuterium kinetic isotope effects and oxidation of substrate analogs

Cyclooxygenases (COX) and 8R-dioxygenase (8R-DOX) activities of linoleate diol synthases (LDS) are homologous heme-dependent enzymes that oxygenate fatty acids by a tyrosyl radical-mediated hydrogen abstraction and antarafacial insertion of O₂. Soybean lipoxygenase-1 (sLOX-1) contains non-heme iron and oxidizes 18:2n ? 6 with a large deuterium kinetic isotope effect (D-KIE). The aim of the present work was to obtain further mechanistic insight into the action of these enzymes by using a series of n ? 6 and n ? 9 fatty acids and by analysis of D-KIE. COX-1 oxidized C₂₀ and C₁₈ fatty acids in the following order of rates: 20:2n ? 6 > 20:1n ? 6 > 20:3n ? 9 > 20:1n ? 9 and 18:3n ? 3 ≥ 18:2n ? 6 > 18:1n ? 6. 18:2n ? 6 and its geometrical isomer (9E,12Z)18:2 were both mainly oxygenated at C-9 by COX-1, but the 9Z,12E isomer was mostly oxygenated at C-13. A cis-configured double bond in the n ? 6 position therefore seems important for substrate positioning. 8R-DOX oxidized (9Z,12E)18:2 at C-8 in analogy with 18:2n ? 6, but the 9E,12Z isomer was mainly subject to hydrogen abstraction at C-11 and oxygen insertion at C-9 by 8R-DOX of 5,8-LDS. sLOX-1 and 13R-MnLOX oxidized [11S-²H]18:2n ? 6 with similar D-KIE (~ 53), which implies that the catalytic metals did not alter the D-KIE. Oxygenation of 18:2n ? 6 by COX-1 and COX-2 took place with a D-KIE of 3–5 as probed by incubations of [11,11-²H₂]- and [11S-²H]18:2n ? 6. In contrast, the more energetically demanding hydrogen abstractions of the allylic carbons of 20:1n ? 6 by COX-1 and 18:1n ? 9 by 8R-DOX were both accompanied by large D-KIE (> 20).     

Statin therapy influences endothelial cell morphology and F-actin cytoskeleton structure when exposed to static and laminar shear stress conditions

AimsTo determine how statin drugs (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) affect endothelial cell (EC) shape and F-actin cytoskeleton arrangement in the presence of physiologically relevant wall shear stress (WSS) of 12.5 dyn/cm².Main methodsHuman abdominal aortic endothelial cells (HAAECs) were cultured to a confluent monolayer within three dimensional tissue culture models and presheared for 6 h at 12.5 dyn/cm² within a continuous flow loop. Statins were added to the perfusion media and the perfusion was continued for a further 24 h. ECs were then analyzed for morphology and F-actin cytoskeleton arrangement using light microscopy and laser scanning confocal microscopy.Key findingsECs became rounded with a significantly higher shape index with the addition of 10 μM simvastatin under both static and flow conditions. F-actin cytoskeleton structure was disorganized and fragmented with statin treatment under static and flow conditions. Neither of these findings were observed with the addition of both simvastatin and 200 μM mevalonate, confirming regulation through the cholesterol biosynthesis pathway.SignificanceEC morphology and F-actin cytoskeleton arrangement are regulated through the cholesterol biosynthesis pathway and are therefore impacted by statin treatment. ECs treated with statins became rounded, which is usually associated with unhealthy cells in regions of the vasculature prone to developing atherosclerotic plaques.     

Arachidonic acid is important for efficient use of light by the microalga Lobosphaera incisa under chilling stress

The oleaginous microalga Lobosphaera incisa (Trebouxiophyceae, Chlorophyta) contains arachidonic acid (ARA, 20:4 n − 6) in all membrane glycerolipids and in the storage lipid triacylglycerol. The optimal growth temperature of the wild-type (WT) strain is 25 °C; chilling temperatures (≤ 15 °C) slow its growth. This effect is more pronounced in the delta-5-desaturase ARA-deficient mutant P127, in which ARA is replaced with dihomo-γ-linolenic acid (DGLA, 20:3 n − 6). In nutrient-replete cells grown at 25 °C, the major chloroplast lipid monogalactosylglycerol (MGDG) was dominated by C18/C16 species in both strains. Yet ARA constituted over 10% of the total fatty acids in the WT MGDG as a component of C20/C18 and C20/C20 species, whereas DGLA was only a minor component of MGDG in P127. Both strains increased the percentage of 18:3 n − 3 in membrane lipids under chilling temperatures. The temperature downshift led to a dramatic increase in triacylglycerol at the expense of chloroplast lipids. WT and P127 showed a similarly high photochemical quantum yield of photosystem II, whereas non-photochemical quenching (NPQ) and violaxanthin de-epoxidation were drastically higher in P127, especially at 15 °C. Fluorescence anisotropy measurements indicated that ARA-containing MGDG might contribute to sustaining chloroplast membrane fluidity upon dropping to the chilling temperature. We hypothesize that conformational changes in chloroplast membranes and increased rigidity of the ARA-deficient MGDG of P127 at chilling temperatures are not compensated by trienoic fatty acids. This might ‘lock’ violaxanthin de-epoxidase in the activated state causing high constitutive NPQ and alleviate the risk of photodamage under chilling conditions in the mutant.     

Chemical compositions and characteristics of organic compounds in propolis from Yemen

Propolis is a gummy material made by honeybees for protecting their hives from bacteria and fungi. The main objective of this study is to determine the chemical compositions and concentrations of organic compounds in the extractable organic matter (EOM) of propolis samples collected from four different regions in Yemen. The propolis samples were extracted with a mixture of dichloromethane and methanol and analyzed by gas chromatography–mass spectrometry (GC–MS). The results showed that the total extract yields ranged from 34% to 67% (mean = 55.5 ± 12.4%). The major compounds were triterpenoids (254 ± 188 mg g⁻¹, mainly α-, β-amyryl and dammaradienyl acetates), n-alkenes (145 ± 89 mg g⁻¹), n-alkanes (65 ± 29 mg g⁻¹), n-alkanoic acids (40 ± 26 mg g⁻¹), long chain wax esters (38 ± 25 mg g⁻¹), n-alkanols (8 ± 3 mg g⁻¹) and methyl n-alkanoates (6 ± 4 mg g⁻¹). The variation in the propolis chemical compositions is apparently related to the different plant sources. The compounds of these propolis samples indicate that they are potential sources of natural bio-active compounds for biological and pharmacological applications.     

Nitrite inhibition and intermediates effects on Anammox bacteria: A batch-scale experimental study

A batch test procedure, based on manometric measurements, was used to study the Anammox process, in particular the inhibition due to nitrite and the effects of hydroxylamine and hydrazine, indicated as possible intermediates of the process. The maximum nitrite removal rate (MNRR) was measured. The method showed good reliability with a standard error of 4.5 ± 3.3% (n: 41). All the tests were carried out on samples taken from a pilot plant with Anammox suspended biomass. The tests were used also to monitor the reactor activity. By testing different spiked additions of nitrite (10–75 mg NO₂⁻-N L⁻¹), a short-term inhibition, with more than 25% MNRR decrease, was found at concentrations higher than 60 mg NO₂⁻-N L⁻¹. Repeated additions of nitrite higher than 30 mg NO₂⁻-N L⁻¹ caused losses of activity. After a complete loss of activity, spiked additions of hydroxylamine (30 mg N L⁻¹ in total) determined a 20% permanent recovery. Low amounts of the intermediates (1–3 mg N L⁻¹) applied on partially inhibited samples and uninhibited samples produced temporary increases in activity up to 50% and 30%, respectively.     

Non-phytin phosphorus requirements of commercial broilers and White Leghorn layers

Experiments were conducted to determine the optimum requirements of non-phytin phosphorus (NPP) in commercial broilers and White Leghorn layers. Five levels of NPP (2.5, 3.0, 3.5, 4.0 and 4.5 g kg⁻¹ diet) were tested to assess the NPP requirement of commercial broilers (3–30 days of age) fed maize–soya diets containing 10 g Ca kg⁻¹. Each level of NPP was fed to quadruplicate groups of ten chicks each. Inclusion of graded levels of NPP significantly (P < 0.01) influenced body weight gain, feed intake, tibia ash content, phosphorus content in serum, tibia ash and phosphorus retention. The predicted NPP requirements for body weight gain, P content in serum and tibia ash were 4.4, 4.48 and 4.1 g kg⁻¹ diet, respectively. The NPP requirement for tibia ash was the highest (7.4 g kg⁻¹ diet). Similarly, four levels of NPP (2.0, 2.5, 3.0 and 3.5 g kg⁻¹ diet) were tested with maize–soya diets containing 35 g Ca kg⁻¹ for White Leghorn layers (266–350 days of age). Each diet was tested on four groups of 12 hens in each. Egg production was not influenced by the variation in dietary NPP levels. The predicted NPP requirements for better egg weight and shell thickness were 2.6 and 2.4 g kg⁻¹ diet, respectively, while for the serum inorganic P level the value was 3.42 g kg⁻¹ diet. Therefore, it can be concluded that commercial broilers need about 4.4 g NPP kg⁻¹ diet for better performance, whereas, White Leghorn layers need not more than 2.0 g NPP kg⁻¹ diet for better egg production. However, layers require 2.6 g NPP kg⁻¹ diet to produce eggs with better egg size and shell quality.     

Improved production of lycopene and β-carotene by Blakeslea trispora with oxygen-vectors

Lycopene and β-carotene production were increased when oxygen-vectors, n-hexane and n-dodecane, were added to cultures of Blakeslea trispora because of the enhanced dissolved oxygen concentrations. With 1% (v/v) n-hexane or n-dodecane added in the medium, lycopene production was 51% or 78% higher and β-carotene production was 44% or 65% higher than that of the control, respectively. The highest lycopene and β-carotene production, 533 mg l⁻¹and 596 mg l⁻¹, were obtained when 1% (v/v) n-dodecane and 0.1% (w/v) Span 20 were added together, which were 2.1-fold and 1.8-fold of the control, respectively.     

Preventive effect of rikkunshito on gastric motor function inhibited by l-dopa in rats

We previously reported that ghrelin prevented l-dopa (LD)-induced inhibition of gastric emptying (GE) of a non-nutrient solution in rats. Parkinson's disease treatment involves the combined administration of l-dopa with the enzyme l-amino acid decarboxylase inhibitor, carbidopa (CD) to reduce peripheral formation of dopamine. We investigated the effect LD/CD given orogastrically (og) on GE of a non-nutrient or nutrient meal and whether og pretreatment with rikkunshito, a kampo medicine clinically used to treat gastroparesis, influenced LD/CD effect on GE and postprandial antral and duodenal motility in conscious rats. LD/CD (20/2 mg kg⁻¹) decreased significantly GE to 26.3 ± 6.0% compared to 61.2 ± 3.2% in og vehicle monitored 20-min after a non-nutrient meal and to 41.9 ± 5.8% compared to 72.9 ± 5.2% in og vehicle monitored 60 min after a nutrient meal. Rikkunshito (0.5 or 1.0 g kg⁻¹) reduced the LD/CD (20/2 mg kg⁻¹) inhibition of GE of non-nutrient meal (36.9 ± 7.4% and 46.6 ± 4.8% respectively vs. 12.1 ± 7.4% in og vehicle plus LD/CD) while having no effect alone (56.6 ± 8.5%). The ghrelin antagonist, [d-Lys³]-GHRP-6 (1 mg kg⁻¹) injected intraperitoneally partially reversed rikkunshito preventive effect on LD/CD-inhibited GE. Rikkunshito (1.0 g kg⁻¹) blocked LD/CD (20/2 mg kg⁻¹)-induced delayed GE of a nutrient meal and the reduction of postprandial antral motility. In 6-hydroxydopamine-induced Parkinson's disease rat model, rikkunshito (1.0 g kg⁻¹, og) also prevented LD/CD-inhibited gastric emptying of a nutrient meal and enhanced fasting plasma levels of acylated ghrelin. These data indicate that oral rikkunshito alleviates the delayed GE induced by LD/CD in naïve and PD rat model in part through ghrelin-related mechanisms.     

Hexane biodegradation in two-liquid phase bioreactors: High-performance operation based on the use of hydrophobic biomass

An innovative operation mode in two-liquid phase bioreactors (TLPB) for the treatment of volatile organic compounds (VOC) was investigated. This mode was based on confining the biocatalytic activity exclusively in the non-aqueous phase (NAP) by using hydrophobic microorganisms. The TLPB was implemented in a 2.5 L stirred tank reactor using 10% (v/v) of silicone oil as NAP and hexane as model VOC. A stable elimination capacity (EC) of 21.0 ± 2.5 g m⁻³ h⁻¹ (corresponding to a removal efficiency of 80%) was recorded for 26 days. The accumulation of inhibitory metabolites resulted in drastic drops in the elimination capacity (EC) and an unstable performance of the system, hexanol being identified as potential inhibitory metabolite. Aqueous culture broth exchange by fresh mineral salt medium at a dilution rate of 0.2 day⁻¹ allowed maintaining a high and sustained VOC removal performance. Dissolved oxygen concentration measurements revealed that the oxidative metabolism was strongly stimulated by the aqueous broth exchange. The temporary blockage of the gas/water/NAP transfer pathway for O₂ highlighted the paramount role of this pathway on the performance of the TLPB based on hydrophobic microorganisms.     

Genotypic variability within Tunisian grapevine varieties (Vitis vinifera L.) facing bicarbonate-induced iron deficiency

Morpho-physiological responses to bicarbonate-induced Fe deficiency were investigated in five Vitis vinifera L. Tunisian varieties (Khamri, Blanc3, Arich Dressé, Beldi, and Balta4). One-month-old woody cuttings were cultivated for 85 days on a free calcareous soil irrigated with tap water containing increasing bicarbonate levels (0, 4, 8, 12, and 16 mM NaHCO₃). After this screening, a second experiment compared root biochemical responses of two contrasting genotypes (tolerant-sensitive) dealing with bicarbonate-induced iron deprivation (20 μM Fe ± 10 mM HCO₃⁻) for 75 days. Using morpho-physiological criteria, grapevine tolerance to HCO₃⁻-induced Fe shortage appeared to be genotype-dependent: Balta4 and Beldi varieties showed the highest leaf-chlorosis score (especially at the extreme HCO₃⁻ levels), in contrast to Khamri variety. Growth parameters (shoot height, total leaf area, leaf number, and biomass production) as well as juvenile leaf chlorophyll content were also differently affected depending on both genotype and bicarbonate dose. At 16 mM HCO₃⁻, Khamri was the less sensitive variety, contrasting with Balta4. On the other hand, chlorophyll content correlated positively with HCl-extractible Fe content of the juvenile leaves, suggesting that the grapevine response to iron deficiency may partly depend on to the plant ability to adequately supply young leaves with this element. Root biochemical responses revealed a relatively higher root acidification capacity in Khamri (tolerant) under Fe-deficiency while no significant changes occurred in Balta4 (sensitive). In addition, Fe(III)-reductase and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activities were strongly stimulated by Fe-deficiency in Khamri, while remaining constant in Balta4. These findings suggest that biochemical parameters may constitute reliable criteria for the selection of tolerant grapevine genotypes to iron chlorosis.     

Estimation of the phosphorus requirement of weanling pigs fed supplemental phytase

Studies were conducted with crossbred weanling pigs to determine the level of phosphorus needed to be fed when a maize–soyabean meal–whey diet was supplemented with exogenous phytase (Natuphos™). In Trial 1, phytase was added at 1200 phytase units (PTU) kg⁻¹ as phosphorus decreased. The control diet in Phase I (0–14 days) contained 7.3 g kg⁻¹ phosphorus and in Phase II (14–28 days) contained 6.5 g kg⁻¹ phosphorus. Dietary phosphorus was calculated to decrease by 0.8, 1.6 or 2.4 g kg⁻¹ when phytase was supplemented. Chromic oxide was added for estimation of apparent absorption of phosphorus. Performance was optimum when 5.7 and 4.8 g kg⁻¹ phosphorus (analysed levels) were fed with 1200 PTU kg⁻¹ phytase in Phases I and II, respectively. The lowest dietary phosphorus levels did not reduce performance for the overall 28-day period. Apparent phosphorus digestibility was increased by phytase in Phase I when 5.7 g kg⁻¹ phosphorus was fed compared to the control diet and in Phase II when 6.0 g kg⁻¹ phosphorus was fed with phytase. Faecal phosphorus excretion decreased in both phases as dietary phosphorus decreased. Faecal phosphorus excretion was minimized at the lowest phosphorus level with no decrease in performance. The estimated requirement for dietary phosphorus, as determined by the NLIN procedure, is 5.0 g kg⁻¹ in Phase I and 4.3 g kg⁻¹ in Phase II when 1200 PTU kg⁻¹ is used. In Trial 2, phytase was supplemented at 500 PTU kg⁻¹ when phosphorus was decreased in the diet. The control diet contained 6.6 and 6.0 g kg⁻¹ phosphorus in Phases I and II, respectively, and phosphorus was calculated to decrease by 0.5, 1.0, 1.5, or 2.0 g kg⁻¹ when phytase was added. Daily gain decreased when 5.0 g kg⁻¹ phosphorus was fed in Phase I and when 4.6 or 4.2 g kg⁻¹ (analysed levels) phosphorus was fed in Phase II with 500 PTU kg⁻¹. Faecal phosphorus excretion decreased as dietary phosphorus decreased, but there were no treatment effects on apparent phosphorus digestibility. The dietary phosphorus requirement was estimated to be 5.7 and 5.0 g kg⁻¹ in Phases I and II, respectively, when phytase is fed at 500 PTU kg⁻¹. At the present recommendation of 500 PTU kg⁻¹ in starter feed, phosphorus can be decreased by 0.10 g kg⁻¹. However, higher levels of phytase are needed to actually increase apparent phosphorus digestibility.