Microbial Oxidation of sec-Alcohols to Ketones

The major anthocyanin compound in buckwheat sprouts was determined to be cyanidin 3-O-rutinoside (C3R), based on HPLC data and MS/MS spectra. Investigation of the content of phenolic compounds in commercial buckwheat sprouts indicated that hypocotyls are abundant in C3R and rutin, whereas all of the detected flavonoids are abundant in cotyledons. The superoxide anion radical-scavenging activities (SOD-like activities) of phenolic compounds in buckwheat sprouts and their contents indicated that rutin, isoorientin, and orientin contributed mainly to the SOD-like activity of the extract from buckwheat sprouts. In contrast, the contribution of C3R was substantially lower than that of flavonoids.     

Phospholipid:diacylglycerol acyltransferase‐mediated triacylglycerol biosynthesis is crucial for protection against fatty acid‐induced cell death in growing tissues of Arabidopsis

Phospholipid:diacylglycerol acyltransferase (PDAT) and diacylglycerol:acyl CoA acyltransferase play overlapping roles in triacylglycerol (TAG) assembly in Arabidopsis, and are essential for seed and pollen development, but the functional importance of PDAT in vegetative tissues remains largely unknown. Taking advantage of the Arabidopsis tgd1–1 mutant that accumulates oil in vegetative tissues, we demonstrate here that PDAT1 is crucial for TAG biosynthesis in growing tissues. We show that disruption of PDAT1 in the tgd1–1 mutant background causes serious growth retardation, gametophytic defects and premature cell death in developing leaves. Lipid analysis data indicated that knockout of PDAT1 results in increases in the levels of free fatty acids (FFAs) and diacylglycerol. In vivo ¹⁴C‐acetate labeling experiments showed that, compared with wild‐type, tgd1–1 exhibits a 3.8‐fold higher rate of fatty acid synthesis (FAS), which is unaffected by disruption or over‐expression of PDAT1, indicating a lack of feedback regulation of FAS in tgd1–1. We also show that detached leaves of both pdat1–2 and tgd1–1 pdat1–2 display increased sensitivity to FFA but not to diacylglycerol. Taken together, our results reveal a critical role for PDAT1 in mediating TAG synthesis and thereby protecting against FFA‐induced cell death in fast‐growing tissues of plants.     

光周期对苦荞芽菜生长与品质的影响

苦荞(Fagopyrum tataricum)芽菜是一种新兴健康食品,该研究通过不同光周期(0、4、8、12、16和20 h·d~(-1))处理苦荞芽菜,测定其生物量、叶绿素和主要营养成分的含量以及黄酮合成相关基因的表达水平,以明确不同光周期对苦荞芽菜品质的影响机理。结果表明:(1)随着苦荞芽菜生长时间的增加,芽菜生物量总体呈上升趋势,16 h·d~(-1)光周期时芽菜生长状态最好,鲜重最大。(2)不同光周期对苦荞芽菜各营养成分含量的影响有所差异,可溶性糖含量在芽菜萌发第2天开始逐渐下降,10 d后含量仅为第2天的13.5%～14.5%;花青素含量在芽菜萌发第2天光周期为12 h·d~(-1)时最高(2.16 mg/g),不同光周期处理4～10 d后均显示出降低趋势;芦丁是苦荞芽菜中主要的黄酮类化合物,芦丁含量在不同光周期处理后有所变化且适当的光照有利于其合成和积累,并在处理第4天光周期为16 h·d~(-1)时含量最高(59.60 mg/g)。(3)qRT-PCR分析表明,不同光周期处理的苦荞芽菜中各黄酮合成相关基因的表达量在第2天最高,随生长周期其表达量均不同程度降低,但黄酮醇合成支路关键酶基因FtFLS1和花青素支路基因FtDFR2表达量上升、且显示出强烈的光诱导特征。研究发现,光周期对苦荞芽菜的生物量影响较大,黑暗有利于胚轴伸长,长光周期(16 h·d~(-1))有利于鲜重的增加;在苦荞萌发过程中,苦荞芽菜可溶性糖、花青素和芦丁含量整体呈减少趋势,可溶性糖和花青素含量均在光周期16 h·d~(-1)处理第2天时最高,芦丁含量在光周期为16 h·d~(-1)处理第4天时最高,建议苦荞芽菜在光周期为16 h·d~(-1)生长4 d时采食。     

Display of adenoregulin with a novel <Emphasis Type="Italic">Pichia pastoris</Emphasis> cell surface display system

Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo 1p with its own secretion signal sequence or the α-factor secretion signal sequence, a polyhistidine (6×His) tag for detection, an enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR.     

Antimicrobial Activity of Rosmarinus officinalis against Oral Pathogens: Relevance of Carnosic Acid and Carnosol

The in vitro inhibitory activity of crude EtOH/H₂O extracts from the leaves and stems of Rosmarinus officinalis L. was evaluated against the following microorganisms responsible for initiating dental caries: Streptococcus mutans, S. salivarius, S. sobrinus, S. mitis, S. sanguinis, and Enterococcus faecalis. Minimum inhibitory concentrations (MIC) were determined with the broth microdilution method. The bioassay‐guided fractionation of the leaf extract, which displayed the higher antibacterial activity than the stem extract, led to the identification of carnosic acid ( 2 ) and carnosol ( 3 ) as the major compounds in the fraction displaying the highest activity, as identified by HPLC analysis. Rosmarinic acid ( 1 ), detected in another fraction, did not display any activity against the selected microorganisms. HPLC Analysis revealed the presence of low amounts of ursolic acid ( 4 ) and oleanolic acid ( 5 ) in the obtained fractions. The results suggest that the antimicrobial activity of the extract from the leaves of R. officinalis may be ascribed mainly to the action of 2 and 3 .     

Functional Compounds in Fermented Buckwheat Sprouts

Fermented buckwheat sprouts (FBS) are used as multifunctional foods. Their production process includes fermentation with lactic acid bacteria. The major strains were found to include Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus pentosus, Lactococcus lactis subsp. lactis, and Pediococcus pentosaceus in an investigation of the lactic acid bacteria. We searched for the functional components, and nicotianamine (NA) and 2″-hydroxynicotianamine (HNA) were identified as angiotensin I-converting enzyme (ACE) inhibitors. NA and HNA increased during fermentation. Indole-3-ethanol was identified as an antioxidant (a SOD active substance), and may have been generated from tryptophan during fermentation because it was not contained in green buckwheat juice. A safety test demonstrated that FBS contained were safe functional food components, showing negative results in buckwheat allergy tests. Any buckwheat allergy substances might have been degraded during the fermentation process.     

Effects of dimerization of the cell‐penetrating peptide Tat analog on antimicrobial activity and mechanism of bactericidal action

The cell‐penetrating peptide Tat (48–60) (GRKKRRQRRRPPQ) derived from HIV‐1 Tat protein showed potent antibacterial activity (MIC: 2–8 µM ). To investigate the effect of dimerization of Tat (48–60) analog, [Tat(W): GRKKRRQRRRPWQ‐NH₂], on antimicrobial activity and mechanism of bactericidal action, its dimeric peptides, di‐Tat(W)‐C and di‐Tat(W)‐K, were synthesized by a disulfide bond linkage and lysine linkage of monomeric Tat(W), respectively. From the viewpoint of a weight basis and the monomer concentration, these dimeric peptides displayed almost similar antimicrobial activity against six bacterial strains tested but acted more rapidly against Staphylococcus aureus on kinetics of bactericidal activity, compared with monomeric Tat(W). Unlike monomeric Tat(W), these dimeric peptides significantly depolarized the cytoplasmic membrane of intact S. aureus cells at MIC and induced dye leakage from bacterial‐membrane‐mimicking egg yolk L ‐α‐phosphatidylethanolamine/egg yolk L ‐α‐phosphatidyl‐DL ‐glycerol (7:3, w/w) vesicles. Furthermore, these dimeric peptides were less effective to translocate across lipid bilayers than monomeric Tat(W). These results indicated that the dimerization of Tat analog induces a partial change in the mode of its bactericidal action from intracellular target mechanism to membrane‐targeting mechanism. Collectively, our designed dimeric Tat peptides with high antimicrobial activity and rapid bactericidal activity appear to be excellent candidates for future development as novel antimicrobial agents. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

UV-B radiation affects flavonoids and fungal colonisation in <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis> and <Emphasis Type="Italic">F. tataricum</Emphasis>

In the present study, we have evaluated the effects of increased UV-B radiation that simulates 17% ozone depletion, on fungal colonisation and concentrations of rutin, catechin and quercetin in common buckwheat (Fagopyrum esculentum) and tartary buckwheat (Fagopyrum tataricum). Induced root growth and reduced shoot:root ratios were seen in both of these buckwheat species after enhanced UV-B radiation. There was specific induction of shoot quercetin concentrations in UV-B-treated common buckwheat, whereas there were no specific responses for flavonoid metabolism in tartary buckwheat. Root colonisation with arbuscular mycorrhizal fungi significantly reduced catechin concentrations in common buckwheat roots, and induced rutin concentrations in tartary buckwheat, but did not affect shoot concentrations of the measured phenolics. Specific UV-B-related reductions in the density of microsclerotia were observed in tartary buckwheat, indicating a mechanism that potentially affects fungus-plant interactions. The data support the hypothesis that responses to enhanced UV-B radiation can be influenced by the plant pre-adaptation properties and related changes in flavonoid metabolism.     

An anthocyanin compound in buckwheat sprouts and its contribution to antioxidant capacity

The major anthocyanin compound in buckwheat sprouts was determined to be cyanidin 3-O-rutinoside (C3R), based on HPLC data and MS/MS spectra. Investigation of the content of phenolic compounds in commercial buckwheat sprouts indicated that hypocotyls are abundant in C3R and rutin, whereas all of the detected flavonoids are abundant in cotyledons. The superoxide anion radical-scavenging activities (SOD-like activities) of phenolic compounds in buckwheat sprouts and their contents indicated that rutin, isoorientin, and orientin contributed mainly to the SOD-like activity of the extract from buckwheat sprouts. In contrast, the contribution of C3R was substantially lower than that of flavonoids.     

HNP-3 enhanced the antimicrobial activity of CIP by promoting ATP efflux from <Emphasis Type="Italic">P. aeruginosa</Emphasis> cells

To establish a novel strategy of P. aeruginosa control, we acquired recombination HNP-3 by gene recombination. Then we examined HNP-3 bio-activities and the influences of antimicrobial peptide on the efflux of ATP. Consequently, we obtained target protein with a molecular mass of 3,000 D consistent with the Anticipation. FIC index of Ciprofloxacin added HNP-3 was less than 0.5 and HNP3 synergistically cooperated with CIP to suppress P. aeruginosa colony formation revealed that there was significant synergy. ATP efflux was however up-regulated by low concentrations of HNP-3, although CIP did not exert any influence on ATP efflux. Conclusively, recombination protein HNP-3 displayed antimicrobial and synergic effects. HNP-3 enhanced the antimicrobial activity of CIP by promoting ATP efflux from P. aeruginosa cells and decreasing efflux of the drugs, which could have useful clinical applications.     

Increased oxidative stress and coenzyme Q10 deficiency in juvenile fibromyalgia: amelioration of hypercholesterolemia and fatigue by ubiquinol-10 supplementation

Abstract

Fibromyalgia (FM) is characterized by generalized pain and chronic fatigue of unknown etiology. To evaluate the role of oxidative stress in this disorder, we measured plasma levels of ubiquinone-10, ubiquinol-10, free cholesterol (FC), cholesterol esters (CE), and free fatty acids (FFA) in patients with juvenile FM (n = 10) and in healthy control subjects (n = 67). Levels of FC and CE were significantly increased in juvenile FM as compared with controls, suggesting the presence of hypercholesterolemia in this disease. However, plasma level of ubiquinol-10 was significantly decreased and the ratio of ubiquinone-10 to total coenzyme Q10 (%CoQ10) was significantly increased in juvenile FM relative to healthy controls, suggesting that FM is associated with coenzyme Q10 deficiency and increased oxidative stress. Moreover, plasma level of FFA was significantly higher and the content of polyunsaturated fatty acids (PUFA) in total FFA was significantly lower in FM than in controls, suggesting increased tissue oxidative damage in juvenile FM. Interestingly, the content of monoenoic acids, such as oleic and palmitoleic acids, was significantly increased in FM relative to controls, probably to compensate for the loss of PUFA. Next, we examined the effect of ubiquinol-10 supplementation (100 mg/day for 12 weeks) in FM patients. This resulted in an increase in coenzyme Q10 levels and a decrease in %CoQ10. No changes were observed in FFA levels or their composition. However, plasma levels of FC and CE significantly decreased and the ratio of FC to CE also significantly decreased, suggesting that ubiquinol-10 supplementation improved cholesterol metabolism. Ubiquinol-10 supplementation also improved chronic fatigue scores as measured by the Chalder Fatigue Scale.     

Characterization of the lipolytic pathways that mediate free fatty acid release during Fas/CD95-induced apoptosis

We have undertaken a study to characterize the lipolytic pathway responsible for the generation of free fatty acids (FFA) during Fas/CD95-induced apoptosis in Jurkat cells. It was initially shown that the cellular lipid fraction that suffered the major quantitative decrease during Fas-induced apoptosis was that of phosphatidylcholine (PC). In addition, the secretion of palmitic acid-derived FFA was largely prevented by D609, an inhibitor of PC-specific phospholipase C (PC-PLC) and also by the diacylglycerol lipase (DAGL) inhibitor RHC-80267, suggesting that the secretion of these FFA during Fas-induced apoptosis is mediated by the generation of DAG by a PC-PLC activity and, sequentially, by a 1-DAGL activity which generates the FFA from its sn-1 position. The endocannabinoid 2-arachidonoyl glycerol (2-AG) should be generated as a sub-product of this pathway, but it did not accumulate inside the cells nor was secreted into the supernatant. Interestingly, the complete inhibition of free AA secretion during Fas-induced apoptosis was only achieved by using the AA trifluoromethylketone, which not only inhibits all types of phospholipase-A₂ (PLA₂) activities, but also the described lytic activities on 2-AG. Using a combination of RHC-80267 and the iPLA₂-specific inhibitor bromoenol lactone, it was shown that the DAGL pathway also cooperates with iPLA₂ in the generation of free arachidonate.     

Enzymatic Characteristics of UDP-sulfoquinovose: Diacylglycerol Sulfoquinovosyltransferase from Chloroplast Envelopes*

Envelope membranes from chloroplasts contain UDP-sulfoquinovose: diacylglycerol sulfoquinovosyltransferase which catalyses the final step in sulfolipid assembly. In situ produced diacylglycerol served as radioactive acceptor to measure enzymatic activity. With this assay, several enzymatic parameters were investigated. The enzyme, which has maximal activity at pH 7.5, was stimulated by magnesium ions due to a decrease of the K_m for uridine 5′-diphospho-sulfoquinovose from 80 pM (no magnesium) to 10 μM (5 mM magnesium). This stimulation had a K_m of 0.7 mM magnesium and may be relevant in light/dark modulation of the enzymatic activity. The lower efficiency of guanosine 5′-diphospho-sulfoquinovose observed before can be ascribed to a higher K_m of this sugar nucleotide (400 μM). Under optimized and linearized conditions the sulfoquinovosyltransferase displayed about 10% of the activity of the UDP-galactose: diacylglycerol galactosyltransferase which competes in the same membrane system for diacylglycerols. Addition of acidic lipids, such as sulfolipid and phosphatidylglycerol, to envelope membranes resulted in an inhibition of the sulfoquinovosyltransferase, whereas the galactosyltransferase was not affected. In vivo this may contribute to an adjustment of the sulfolipid proportion in plastid membranes. In contrast to the galactosyltransferase the sulfoquinovosyltransferase did not discriminate against the dipalmitoyl molecular species of diacylglycerol when offered together with the oleoyl-palmitoyl species. Under conditions when oleoyl-palmitoyl-and dipalmitoyl-diacylglycerols were synthesized with concurrent conversion to monogalactosyl and sulfoquinovosyl diacylglycerol, the sulfolipid was highly enriched in the fully saturated species. This may explain the occurrence of dipalmitoyl species in sulfolipids, as found in many plants.     

Extracellular Ionic Locks Determine Variation in Constitutive Activity and Ligand Potency between Species Orthologs of the Free Fatty Acid Receptors FFA2 and FFA3

Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein-coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct rank-order of potency such that acetate (C2) has been described as FFA2-selective, whereas propionate (C3) is non-selective. Although C2 was confirmed to be selective for human FFA2 over FFA3, this ligand was not selective between the mouse orthologs. Moreover, although C3 was indeed not selective between the human orthologs, it displayed clear selectivity for mouse FFA3 over mouse FFA2. This altered selectivity to C2 and C3 resulted from broad differences in SCFAs potency at the mouse orthologs. In studies to define the molecular basis for these observations, marked variation in ligand-independent constitutive activity was identified using a [³⁵S]GTPγS assay. The orthologs with higher potency for the SCFAs, human FFA2 and mouse FFA3, displayed high constitutive activity in this assay, whereas the orthologs with lower potency for the agonist ligands, mouse FFA2 and human FFA3, did not. Sequence alignments of the second extracellular loop identified single negatively charged residues in FFA2 and FFA3 not conserved between species and predicted to form ionic lock interactions with arginine residues within the FFA2 or FFA3 agonist binding pocket to regulate constitutive activity and SCFA potency. Reciprocal mutation of these residues between species orthologs resulted in the induction (or repression) of constitutive activity and in most cases also yielded corresponding changes in SCFA potency.     

Synthesis and Antimicrobial Evaluation of Novel Chiral 2‐Amino‐4,5,6,7‐tetrahydrothieno[2,3‐c]pyridine Derivatives

New N‐substituted‐2‐amino‐4,5,6,7‐tetrahydrothieno[2,3‐c]pyridine derivatives were synthesized employing a convenient one‐pot three‐component method and their structures were characterized by ¹H‐NMR and single crystal X‐ray diffraction analysis. All the synthesized compounds were in vitro screened for antimicrobial activity against Gram‐positive (Sarcina lutea) and Gram‐negative bacteria (Escherichia coli). In this work, we introduced a chiral residue on the tetrahydropyridine nitrogen, the hitherto the less investigated position on this pharmacophore in order to explore the effect. The antibacterial results showed that the synthesized compounds were active only against Gram‐positive bacteria and the (R)‐enantiomers displayed a greater antimicrobial potency than their (S)‐counterparts. The structure–activity relationship here investigated may provide some interesting clues for future development of tetrahydrothienopyridine derivatives with higher antimicrobial activity.     

Light emitting diodes increase phenolics of buckwheat (Fagopyrum esculentum) sprouts

Abstract

Four types of light emitting diodes (LEDs) were used in three combinations (all blue [BBB], red: blue: far-red [RBFr]=4:1:1 and red: green: blue [RGB]=4:1:1) and seven irradiation cycles (12 h/12 h photoperiod, 1 Hz, 10 Hz, 100 Hz, 1 KHz, 10 KHz and 100 KHz) were applied to examine their effect on growth, stem color and phenolic compounds (homoorientin, orientin, rutin, vitexin and isovitexin) in buckwheat (Fagopyrum esculentum cv. Great Ruby) sprouts. With a few exceptions, among all the combinations and irradiation cycles of LEDs, the Great Ruby cultivar showed significant differences in total height and dry weight of sprouts. The study revealed that there was a direct effect of different combination and irradiation cycles of LEDs on sprout height and dry weight; and among the combinations, RBFr and BBB had more positive effect than RGB. Increasing irradiation cycles markedly decreased the red color of stalks of F. esculentum cv. Great Ruby sprouts, with no significant effect on leaf color. In sprouts, the rutin content of leaves and stalks was higher at 12 h photoperiod and the amount in leaves was 5–7% higher than that of stalks in all combinations and irradiation cycles of LEDs. Among polyphenols, the rutin content in stalks was 1.45–2.97 mg g^?1 dry weight and the maximum amount of rutin was obtained from RGB combination of LEDs. In leaves, higher amounts of homoorientin, orientin and vitexin+isovitexin were obtained from the RGB combination and there were significant differences between RGB and other two combinations of LEDs. On the other hand, in between the irradiation cycles used in the study, there were no significant differences for other polyphenols in leaves and stalks of buckwheat sprouts.     

Effect of Farnesol on Planktonic and Biofilm Cells of <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis>

Staphylococcus epidermidis is now amongst the most important pathogenic agents responsible for bloodstream nosocomial infections and for biofilm formation on indwelling medical devices. Its increasing resistance to common antibiotics is a challenge for the development of new antimicrobial agents. Accordingly, the goal of this study was to evaluate the effect of farnesol, a natural sesquiterpenoid, on Staphylococcus epidermidis planktonic and biofilm cells. Farnesol displayed a significant inhibitory effect on planktonic cells. Small concentrations (100 μM) were sufficient to exhibit antibacterial effect on these cells. In biofilm cells the effect of farnesol was not so pronounced and it seems to be strongly dependent on the cells metabolic activity and amount of matrix. Interestingly, the effect of farnesol at 200 μM was similar to the effect of vancomycin at peak serum concentration either in planktonic or biofilm cells. Overall, the results indicate a potential antibacterial effect of farnesol against S. epidermidis, and therefore the possible action of this molecule on the prevention of S. epidermidis related infections.     

Impact of flowering buckwheat on Lepidopteran cabbage pests and their parasitoids at two spatial scales

We assessed the potential of annual buckwheat, Fagopyrum esculentum Moench, to lead to improved parasitism of lepidopteran cabbage pests over four years. Pest, parasitism, and hyperparasitism rates were monitored in replicated cabbage plots (12 × 20 m) with or without 3 m wide buckwheat borders from 2000 to 2003. Floral borders did not significantly increase egg, larval, or pupal densities of cabbage looper, Trichoplusia ni (Hübner), imported cabbageworm, Pieris rapae (L.), or diamondback moth, Plutella xylostella (L.). Buckwheat increased parasitism rates by Voria ruralis (Fallen) on T. ni larvae and Cotesia rubecula (Marshall) on P. rapaelarvae over four years. Parasitism by Diadegma insulare (Cresson) on P. xylostella larvae was higher in buckwheat than control plots in the first year, and parasitism by Euplectrus plathypenae (Howard) on T. ni larvae was lower in buckwheat than control plots in the second year. The hyperparasitoid Conura side (Walker) attacked D. insulare all four years, but buckwheat did not affect hyperparasitism rates. The effect of spatial scale on pest densities and parasitism in 2001 was evaluated by comparing plots separated at least 67 m (nearby) versus 800 m apart (isolated). T. ni pupae and P. rapae eggs and pupae were more abundant in plots in closer proximity, whereas P. xylostella densities did not vary by the spatial separation of plots. Tachinids and Pteromalus puparum (L.) attacked more P. rapae in nearby plots. E. plathypenae responded to the treatment × scale interaction, parasitizing more in control than buckwheat when plots were isolated but not when plots were nearby.     

Regulation of Acetyl CoA Carboxylase and Carnitine Palmitoyl Transferase‐1 in Rat Adipocytes

Objective: Acetyl CoA carboxylase (ACC) is a key enzyme in energy balance. It controls the synthesis of malonyl‐CoA, an allosteric inhibitor of carnitine palmitoyltransferase‐1 (CPT‐I). CPT‐I is the gatekeeper of free fatty acid (FFA) oxidation. To test the hypothesis that both enzymes play critical roles in regulation of FFA partitioning in adipocytes, we compared enzyme mRNA expression and specific activity from fed, fasted, and diabetic rats. Research Methods and Procedures: Direct effects of nutritional state, insulin, and FFAs on CPT‐I and ACC mRNA expression were assessed in adipocytes, liver, and cultured adipose tissue explants. We also determined FFA partitioning in adipocytes from donors exposed to different nutritional conditions. Results: CPT‐I mRNA and activity decreased in adipocytes but increased in liver in response to fasting. ACC mRNA and activity decreased in both adipocytes and liver during fasting. These changes were not caused directly by fasting‐associated changes in plasma insulin and FFA concentrations because insulin suppressed CPT‐I mRNA and did not affect ACC mRNA in vitro, whereas exogenous oleate had no effect on either. Despite the decrease in adipocyte CPT‐I mRNA and specific activity, CO₂ production from endogenous FFAs increased, suggesting increased FFA transport through CPT‐I for β‐oxidation. Discussion: Stimulation of FFA transport through CPT‐I occurs in both tissues, but CPT‐I mRNA and specific activity correlate with FFA transport in liver and not in adipocytes. We conclude that the mechanism responsible for increasing FFA oxidation in adipose tissue during fasting involves mainly allosteric regulation, whereas altered gene expression may play a central role in the liver.     

Structure‐activity relationship studies of shortened analogues of the antimicrobial peptide EeCentrocin 1 from the sea urchin Echinus esculentus

EeCentrocin 1 is a potent antimicrobial peptide isolated from the marine sea urchin Echinus esculentus. The peptide has a hetero‐dimeric structure with the antimicrobial activity confined in its largest monomer, the heavy chain (HC), encompassing 30 amino acid residues. The aim of the present study was to develop a shorter drug lead peptide using the heavy chain of EeCentrocin 1 as a starting scaffold and to perform a structure‐activity relationship study with sequence modifications to optimize antimicrobial activity. The experiments consisted of 1) truncation of the heavy chain, 2) replacement of amino acids unfavourable for in vitro antimicrobial activity, and 3) an alanine scan experiment on the truncated and modified heavy chain sequence to identify essential residues for antimicrobial activity. The heavy chain of EeCentrocin 1 was truncated to less than half its initial size, retaining most of its original antimicrobial activity. The truncated and optimized lead peptide ( P6 ) consisted of the 12 N‐terminal amino acid residues from the original EeCentrocin 1 HC sequence and was modified by two amino acid replacements and a C‐terminal amidation. Results from the alanine scan indicated that the generated lead peptide ( P6 ) contained the optimal sequence for antibacterial activity, in which none of the alanine scan peptides could surpass its antimicrobial activity. The lead peptide ( P6 ) was also superior in antifungal activity compared to the other peptides prepared and showed minimal inhibitory concentrations (MICs) in the low micromolar range. In addition, the lead peptide ( P6 ) displayed minor haemolytic and no cytotoxic activity, making it a promising lead for further antimicrobial drug development.