Identification and characterization of a fatty acyl reductase from a Spodoptera littoralis female gland involved in pheromone biosynthesis

Fatty acyl‐CoA reductases (FARs), the enzymes that catalyse reduction of a fatty acyl‐CoA to the corresponding alcohol in insect pheromone biosynthesis, are postulated to play an important role in determining the proportion of each component in the pheromone blend. For the first time, we have isolated and characterized from the Egyptian cotton leaf worm Spodoptera littoralis (Lepidoptera: Noctuidae) a FAR cDNA (Slit‐FAR1), which appeared to be expressed only in the pheromone gland and was undetectable in other female tissues, such as fat body, ovaries, wings, legs or thorax. The encoded protein has been successfully expressed in a recombinant system, and the recombinant enzyme is able to produce the intermediate fatty acid alcohols of the pheromone biosynthesis of S. littoralis from the corresponding acyl‐CoA precursors. The kinetic variables Km and Vmax, which have been calculated for each acyl‐CoA pheromone precursor, suggest that in S. littoralis pheromone biosynthesis other biosynthetic enzymes (e.g. desaturases, acetyl transferase) should also contribute to the final ratio of components of the pheromone blend. In a phylogenetic analysis, Slit‐FAR1 appeared grouped in a cluster of other FARs involved in the pheromone biosynthesis of other insects, with little or non‐specificity for the natural pheromone precursors.     

Resveratrol inhibits fatty acid and triacylglycerol synthesis in rat hepatocytes

Background The putative role of resveratrol, a polyphenol present in grapes and other plants, in modulating dislypidemia, thus preventing cardiovascular diseases, is generally based on proliferating cell lines and in vivo studies in different pathological conditions. The aim of the present study was to investigate whether resveratrol plays a role on lipid biosynthesis in rat hepatocytes. Materials and methods The effect of resveratrol on total rate of fatty acid, cholesterol and complex lipid synthesis, assayed by the incorporation of [1‐¹⁴C]acetate into these lipid fractions, was investigated in rat hepatocyte suspensions. Enzyme activities of acetyl‐CoA carboxylase (ACC) and fatty acid synthase (FAS) as well as 3‐hydroxy‐3‐methyl‐glutaryl‐CoA reductase (HMG‐CoA‐R), pace‐setting steps of de novo fatty acid and cholesterol synthesis, respectively, were in situ measured in digitonin‐permeabilized hepatocytes. Results Resveratrol‐treated hepatocytes exhibited a short‐term (30 min) inhibition (IC₅₀ ~25 µm ) of total fatty acid synthesis from [1‐¹⁴C]acetate. Among neosynthesized fatty acids, palmitic acid formation was mainly reduced, thus suggesting that enzymatic step(s) of de novo fatty acid synthesis was affected by resveratrol. In digitonin‐permeabilized hepatocytes, only ACC activity was noticeably reduced, while no change in FAS activity was observed. A noticeable resveratrol‐induced reduction of label incorporation into triacylglycerols was also detected. Conversely, cholesterol synthesis and HMG‐CoA‐R activity were unaffected by resveratrol. Conclusion Results here reported show that in isolated hepatocytes from normal rats a resveratrol‐induced short‐term inhibition of fatty acid and triacylglycerol synthesis occurs. This finding may represent a potential mechanism contributing to the reported hypolipidemic effect of resveratrol.     

Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2.

We produced transgenic mice that express a dominant-positive truncated form of sterol regulatory element-binding protein-2 (SREBP-2) in liver and adipose tissue. The encoded protein lacks the membrane-binding and COOH-terminal regulatory domains, and it is therefore not susceptible to negative regulation by cholesterol. Livers from the transgenic mice showed increases in mRNAs encoding multiple enzymes of cholesterol biosynthesis, the LDL receptor, and fatty acid biosynthesis. The elevations in mRNA for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase were especially marked (13-fold and 75-fold, respectively). As a result, the transgenic livers showed a 28-fold increase in the rate of cholesterol synthesis and a lesser fourfold increase in fatty acid synthesis, as measured by intraperitoneal injection of [3H]water. These results contrast with previously reported effects of dominant-positive SREBP-1a, which activated fatty acid synthesis more than cholesterol synthesis. In adipose tissue of the SREBP-2 transgenics, the mRNAs for cholesterol biosynthetic enzymes were elevated, but the mRNAs for fatty acid biosynthetic enzymes were not. We conclude that SREBP-2 is a relatively selective activator of cholesterol synthesis, as opposed to fatty acid synthesis, in liver and adipose tissue of mice.     

Enzymes catalyzing the early steps of clavulanic acid biosynthesis are encoded by two sets of paralogous genes in Streptomyces clavuligerus

Genes encoding the proteins required for clavulanic acid biosynthesis and for cephamycin biosynthesis are grouped into a "supercluster" in Streptomyces clavuligerus. Nine open reading frames (ORFs) associated with clavulanic acid biosynthesis were located in a 15-kb segment of the supercluster, including six ORFs encoding known biosynthetic enzymes or regulatory proteins, two ORFs that have been reported previously but whose involvement in clavulanic acid biosynthesis is unclear, and one ORF not previously reported. Evidence for the involvement of these ORFs in clavulanic acid production was obtained by generating mutants and showing that all were defective for clavulanic acid production when grown on starch asparagine medium. However, when five of the nine mutants, including mutants defective in known clavulanic acid biosynthetic enzymes, were grown in a soy-based medium, clavulanic acid-producing ability was restored. This ability to produce clavulanic acid when seemingly essential biosynthetic enzymes have been mutated suggests that paralogous genes encoding functionally equivalent proteins exist for each of the five genes but that these paralogues are expressed only in the soy-based medium. The five genes that have paralogues encode proteins involved in the early steps of the pathway common to the biosynthesis of both clavulanic acid and the other clavam metabolites produced by this organism. No evidence was seen for paralogues of the four remaining genes involved in late, clavulanic acid-specific steps in the pathway.     

Development of lacrimal gland spheroids for lacrimal gland tissue regeneration

Severe dry eye syndrome resulting from lacrimal gland (LG) dysfunction can cause blindness, yet treatments remain palliative. In vitro reconstruction of LG tissue could provide a curative treatment. We aimed to combine epithelial cells with endothelial cells and mesenchymal stem cells (MSCs) to form a 3D functional unit. Epithelial cells and MSCs were isolated from porcine LG; endothelial cells were isolated from human foreskin. MSCs were characterised (flow cytometry and differentiation potential assays). All 3 cell types were combined on Matrigel and spheroid formation observed. Spheroids were characterised [immunohistochemistry (IHC) and transmission electron microscopy] and function assessed (β‐hexosaminidase assay). Spheroids were transferred to decellularised jejunum (SIS‐Muc) in dynamic cultures for 1 week before further characterisation. MSCs did not express CD31 but expressed CD44 and CD105 and differentiated towards osteogenic and adipogenic lineages. Spheroids formed on Matrigel within 18 hr, contracting to ~10% of the well area (p < .005). IHC revealed presence of all 3 cells within spheroids. Transmission electron microscopy revealed cell–cell contacts and polarisation at the apical surface. In static cultures, function was increased in spheroids cf. monolayer controls (p < .05) but over 72 hr, spheroid function (p < .05), viability (p < .05), and proliferation decreased, whilst apoptosis increased. On SIS‐Muc under dynamic culture, however, spheroids continued to proliferate to repopulate SIS‐Muc. IHC revealed LG epithelial cells coexpressing pan‐cytokeratin and lysozyme, as well as endothelial cells and MSCs and cells remained capable of responding to carbachol (p < .05). These spheroids could form the basis of a regenerative medicine treatment approach for dry eye syndrome. In vivo studies are required to evaluate this further.     

Microparticle‐associated tissue factor activity,venous thromboembolism and mortality in pancreatic,gastric, colorectal and brain cancer patients

Summary. Background: Tissue factor (TF) expression by tumors contributes to tumor growth. Release of TF‐positive microparticles (MPs) may contribute to venous thromboembolism (VTE). Objectives: To conduct a prospective cohort study to determine whether elevated MP‐associated TF (MP‐TF) activity is predictive of VTE and mortality in four cancer types. Patients/Methods: We determined MP‐TF activity in pancreatic, gastric, colorectal and brain cancer patients. We used a chromogenic endpoint assay for all patients and also a chromogenic kinetic assay for patients with pancreatic and brain cancer. Results: During follow‐up, 12/60 (20%) pancreatic, 6/43 (14%) gastric, 12/126 (10%) colorectal and 19/119 (16%) brain cancer patients developed VTE; 46/60 (77%), 30/43 (70%), 47/126 (37%) and 67/119 (56%), respectively, died. MP‐TF activity levels were highest in pancreatic cancer. We did not find a statistically significant association of MP‐TF activity with the risk of VTE in any of the four cancer types by using two statistical methods. An association of MP‐TF activity with the risk of mortality was detected in pancreatic cancer with the endpoint assay (hazard ratio [HR] 1.8 and 95% confidence interval [CI] 1.4–2.3 per doubling of activity, P < 0.001) and the kinetic assay (HR 1.2, 95% CI 1.1–1.4, P < 0.001); adjustment for type of treatment was not performed. In pancreatic cancer, MP‐TF activity correlated with D‐dimer level (endpoint assay, r = 0.51; chromogenic assay, r = 0.48), and a correlation between assays (r = 0.61) was found. Conclusion: MP‐TF activity was not associated with future VTE in pancreatic, gastric, colorectal and brain cancer. However, we found a strong association of MP‐TF activity with mortality in pancreatic cancer. MP‐TF activity might be reflective of an aggressive pancreatic cancer phenotype.     

Segregation of motor and sensory axons regenerating through bicompartmental tubes by combining extracellular matrix components with neurotrophic factors

Segregation of regenerating motor and sensory axons may be a good strategy to improve selective functionality of regenerative interfaces to provide closed‐loop commands. Provided that extracellular matrix components and neurotrophic factors exert guidance effects on different neuronal populations, we assessed in vivo the potential of separating sensory and motor axons regenerating in a bicompartmental Y‐type tube, with each branch prefilled with an adequate combination of extracellular matrix and neurotrophic factors. The severed rat sciatic nerve was repaired using a bicompartmental tube filled with a collagen matrix enriched with fibronectin (FN) and brain‐derived neurotrophic factor (BDNF) encapsulated in poly‐lactic co‐glycolic acid microspheres (FN + MP.BDNF) in one compartment to preferentially attract motor axons and collagen enriched with laminin (LM) and nerve growth factor (NGF) and neurotrophin‐3 (NT‐3) in microspheres (LM + MP.NGF/NT‐3) in the other compartment for promoting sensory axons regeneration. Control animals were implanted with the same Y‐tube with a collagen matrix with microspheres (MP) containing PBS (Col + MP.PBS). By using retrotracer labelling, we found that LM + MP.NGF/NT‐3 did not attract higher number of regenerated sensory axons compared with controls, and no differences were observed in sensory functional recovery. However, FN + MP.BDNF guided a higher number of regenerating motor axons compared with controls, improving also motor recovery. A small proportion of sensory axons with large soma size, likely proprioceptive neurons, was also attracted to the FN + MP.BDNF compartment. These results demonstrate that muscular axonal guidance can be modulated in vivo by the addition of fibronectin and BDNF.     

Ventriculo–arterial interaction may be assessed by Oscillatory Power Fraction

The rate of energy transfer from the left ventricle to the aorta is viewed in terms of mean power (MP) and total power (TP). The difference between MP and TP is due to the pulsatility of the circulation and is known as oscillatory power (OP). OP is considered the energy spent to accelerate the blood flow. The aim of this study was to investigate the baseline left ventricular oscillatory power fraction (OP/TP) and how this was affected by acute cardiovascular dysfunction and altered preload. Twenty‐eight patients undergoing elective coronary artery bypass graft surgery were included. Before administration of anaesthesia, we simultaneously recorded an arterial pressure curve and instantaneous cardiac outflow with pulsed wave Doppler. Postoperatively, prior to extubation, these measurements were repeated in neutral, Trendelenburg and reverse‐Trendelenburg position. The final measurements were taken on the awake patient the day after the operation. TP is the mean of the instantaneous product of the flow and pressure curves. MP was calculated by multiplying mean arterial pressure with mean cardiac output. The oscillatory power fraction is therefore calculated as (TP‐MP)/TP. The oscillatory power fraction in neutral position decreased from 23% preoperatively to 16% immediately postoperatively (P<0·001) and increased again to 19% the first postoperative day (P = 0·001). The oscillatory power fraction also increased from 16% in neutral to 19% in Trendelenburg (P = 0·001) and decreased comparing to neutral, to 14% in reverse‐Trendelenburg (P = 0·04). The oscillatory power fraction is situation‐dependent and is influenced by both the operation and the altered preload.     

ATP Citrate Lyase Knockdown Induces Growth Arrest and Apoptosis through Different Cell- and Environment-Dependent Mechanisms

ATP citrate lyase (ACLY) is a cytosolic enzyme that catalyzes generation of acetyl-CoA, which is a vital building block for fatty acid, cholesterol, and isoprenoid biosynthesis. ACLY is upregulated in several types of cancer, and its inhibition induces proliferation arrest in certain cancer cells. As ACLY is involved in several pathways, its downregulation may affect multiple processes. Here, we have shown that short hairpin RNA-mediated ACLY silencing in cell lines derived from different types of cancers induces proliferation, cell-cycle arrest, and apoptosis. However, this antiproliferative effect of ACLY knockdown was observed only when cells were cultivated under lipid-reduced growth conditions. Proliferation arrest induced by ACLY silencing was partially rescued by supplementing the media with fatty acids and/or cholesterol. This indicates that the ACLY knockdown-mediated growth arrest might be the result of either fatty acid or cholesterol starvation or both. In the absence of ACLY, the cancer cells displayed elevated expression of sterol regulatory element binding protein-regulated downstream genes involved in de novo fatty acid and cholesterol biosynthesis. Furthermore, ACLY suppression resulted in elevated expression of acyl-CoA synthetase short-chain family member 2 (ACSS2), an enzyme that also produces acetyl-CoA using acetate as a substrate. Acetate supplementation partially rescued the cancer cells from ACLY suppression-induced proliferation arrest. We also observed that the absence of ACLY enhanced ACSS2-dependent lipid synthesis. These findings provide new insights into the role of ACLY in cancer cell growth and give critical information about the effects of ACLY silencing on different pathways. This information is crucial in understanding the possible application of ACLY inhibition in cancer therapeutics. Mol Cancer Ther; 11(9); 1925-35. ?2012 AACR.     

Roles of vitamin A in the regulation of fatty acid synthesis

Dietary macronutrients and micronutrients play important roles in human health. On the other hand, the excessive energy derived from food is stored in the form of triacylglycerol. A variety of dietary and hormonal factors affect this process through the regulation of the activities and expression levels of those key player enzymes involved in fatty acid biosynthesis such as acetyl-CoA carboxylase, fatty acid synthase, fatty acid elongases, and desaturases. As a micronutrient, vitamin A is essential for the health of humans. Recently, vitamin A has been shown to play a role in the regulation of glucose and lipid metabolism. This review summarizes recent research progresses about the roles of vitamin A in fatty acid synthesis. It focuses on the effects of vitamin A on the activities and expression levels of mRNA and proteins of key enzymes for fatty acid synthesis in vitro and in vivo. It appears that vitamin A status and its signaling pathway regulate the expression levels of enzymes involved in fatty acid synthesis. Future research directions are also discussed.     

Influence of exercise order on upper body maximum and submaximal strength gains in trained men

The purpose of this study was to investigate the influence of exercise order on one‐repetition maximum (1‐RM) and ten‐repetition maximum (10‐RM) strength gains after 6 weeks of resistance training (RT) in trained men. Sixteen men were randomly assigned into two groups based on the order of exercises performed during training sessions: a group that performed large muscle group exercises first and progressed to small muscle group exercises (LG‐SM); while a second group performed the opposite sequence and started with small muscle group exercises and progressed to large muscle group exercises (SM‐LG). Four sessions of RT were conducted per week; all exercises were performed for three sets of 8–12 repetitions with 1‐min rest intervals between sets. Maximal and submaximal strength were assessed at baseline and after 6 weeks of RT with 1‐RM and 10‐RM testing for the bench press (BP), lat pulldown (LPD), triceps pulley extension (TE) and biceps curl (BC), respectively. Two‐way ANOVA for the 1‐RM and 10‐RM tests indicated a significant group x time interaction. The 1‐RM values significantly increased for all exercises in both groups (P<0.05), but were not significantly different between groups. However, effect size (ES) data indicated that the LG‐SM group exhibited a greater magnitude of gains (1‐RM and 10‐RM) for the BP and LPD exercises. Conversely, ES indicated that the SM‐LG group exhibited a greater magnitude of gains (1‐RM and 10‐RM) for the TE and BC exercises. In conclusion, the results suggest that upper body movements should be prioritized and performed according to individual needs to maximize maximal and submaximal strength.     

Care provider allocation on admissions to acute mental health wards: The development and validation of the Admission Team Score List

Currently, support tools are lacking to prioritize steps in the care coordination process to enable safe practice and effective clinical pathways in the first phase of acute psychiatric admissions. This study describes the development, validity, and reliability of an acute care coordination support tool, the Admission Team Score List (ATSL). The ATSL assists in care provider allocation during admissions. Face validity and feasibility of the ATSL were tested in 77 acute admissions. Endscores of filled out ATSL's were translated to recommended team compositions. These ATSL team (ATSL‐T) compositions were compared to the actually present team (AP‐T) and the most preferred team (MP‐T) composition in hindsight. Consistency between the ATSL‐T and the MP‐T was substantial; K_w = 0.70, P < 0.001, 95% CI [0.55–0.84]. The consistency between the ATSL‐T and AP‐T was moderate; K_w = 0.43, P < 0.001, 95% CI [0.23–0.62]. The ATSL has an adequate (inter‐rater) reliability; ICC = 0.90, P < 0.001, 95% CI [0.65–0.91]. The ATSL study is an important step to promote safety and efficient care based on care provider allocation, for service users experiencing an acute admission. The ATSL may stimulate structured clinical decision‐making during the hectic process around acute psychiatric admissions.     

Proteomic signature of fatty acid biosynthesis inhibition available for in vivo mechanism-of-action studies

Fatty acid biosynthesis is a promising novel antibiotic target. Two inhibitors of fatty acid biosynthesis, platencin and platensimycin, were recently discovered and their molecular targets identified. Numerous structure-activity relationship studies for both platencin and platensimycin are currently being undertaken. We established a proteomic signature for fatty acid biosynthesis inhibition in Bacillus subtilis using platencin, platensimycin, cerulenin, and triclosan. The induced proteins, FabHA, FabHB, FabF, FabI, PlsX, and PanB, are enzymes involved in fatty acid biosynthesis and thus linked directly to the target pathway. The proteomic signature can now be used to assess the in vivo mechanisms of action of compounds derived from structure-activity relationship programs, as demonstrated for the platensimycin-inspired chromium bioorganometallic PM47. It will further serve as a reference signature for structurally novel natural and synthetic antimicrobial compounds with unknown mechanisms of action. In summary, we described a proteomic signature in B. subtilis consisting of six upregulated proteins that is diagnostic of fatty acid biosynthesis inhibition and thus can be applied to advance antibacterial drug discovery programs.