Revisiting the coupling of fatty acid to phospholipid synthesis in bacteria with FapR regulation

A key aspect in membrane biogenesis is the coordination of fatty acid to phospholipid synthesis rates. In most bacteria, PlsX is the first enzyme of the phosphatidic acid synthesis pathway, the common precursor of all phospholipids. Previously, we proposed that PlsX is a key regulatory point that synchronizes the fatty acid synthase II with phospholipid synthesis in Bacillus subtilis. However, understanding the basis of such coordination mechanism remained a challenge in Gram-positive bacteria. Here, we show that the inhibition of fatty acid and phospholipid synthesis caused by PlsX depletion leads to the accumulation of long-chain acyl-ACPs, the end products of the fatty acid synthase II. Hydrolysis of the acyl-ACP pool by heterologous expression of a cytosolic thioesterase relieves the inhibition of fatty acid synthesis, indicating that acyl-ACPs are feedback inhibitors of this metabolic route. Unexpectedly, inactivation of PlsX triggers a large increase of malonyl-CoA leading to induction of the fap regulon. This finding discards the hypothesis, proposed for B. subtilis and extended to other Gram-positive bacteria, that acyl-ACPs are feedback inhibitors of the acetyl-CoA carboxylase. Finally, we propose that the continuous production of malonyl-CoA during phospholipid synthesis inhibition provides an additional mechanism for fine-tuning the coupling between phospholipid and fatty acid production in bacteria with FapR regulation.     

Synthesis and evaluation of new 1-oxa-8-azaspiro[4.5]decane derivatives as candidate radioligands for sigma-1 receptors

We report the design, synthesis, and evaluation of a series of 1-oxa-8-azaspiro[4.5]decane and 1,5-dioxa-9-azaspiro[5.5]undecane derivatives as selective σ₁ receptor ligands. All seven ligands exhibited nanomolar affinity for σ₁ receptors (K_i(σ₁) = 0.47 – 12.1 nM) and moderate selectivity over σ₂ receptors (K_i(σ₂)/ K_i(σ₁) = 2 – 44). Compound 8, with the best selectivity among these ligands, was selected for radiolabeling and further evaluation. Radioligand [¹⁸F]8 was prepared via nucleophilic ¹⁸F-substitution of the corresponding tosylate precursor, with an overall isolated radiochemical yield of 12–35%, a radiochemical purity of greater than 99%, and molar activity of 94 – 121 GBq/μmol. Biodistribution studies of [¹⁸F]8 in mice demonstrated high initial brain uptake at 2 min. Pretreatment with SA4503 resulted in significantly reduced brain-to-blood ratio (70% − 75% at 30 min). Ex vivo autoradiography in ICR mice demonstrated high accumulation of the radiotracer in σ₁ receptor-rich brain areas. These findings suggest that [¹⁸F]8 could be a lead compound for further structural modifications to develop potential brain imaging agents for σ₁ receptors.     

Impaired G2/M cell cycle arrest induces apoptosis in pyruvate carboxylase knockdown MDA-MB-231 cells

BackgroundPrevious studies showed that suppression of pyruvate carboxylase (PC) expression in highly invasive breast cancer cell line, MDA-MB-231 inhibits cell growth as a consequence of the impaired cellular biosynthesis. However, the precise cellular mechanism underlying this growth restriction is unknown.MethodsWe generated the PC knockdown (PCKD) MDA-MB-231 cells and assessed their phenotypic changes by fluorescence microscopy, proliferation, apoptotic, cell cycle assays and proteomics.ResultsPC knockdown MDA-MB-231 cells had a low percentage of cell viability in association with accumulation of abnormal cells with large or multi-nuclei. Flow cytometric analysis of annexin V-7-AAD positive cells showed that depletion of PC expression triggers apoptosis with the highest rate at day 4. The increased rate of apoptosis is consistent with increased cleavage of procaspase 3 and poly (ADP-Ribose) polymerase. Cell cycle analysis showed that the apoptotic cell death was associated with G2/M arrest, in parallel with marked reduction of cyclin B levels. Proteomic analysis of PCKD cells identified 9 proteins whose expression changes were correlated with the degree of apoptosis and G2/M cell cycle arrest in the PCKD cells. STITCH analysis indicated 3 of 9 candidate proteins, CCT3, CABIN1 and HECTD3, that form interactions with apoptotic and cell cycle signaling networks linking to PC via MgATP.ConclusionsSuppression of PC in MDA-MB-231 cells induces G2/M arrest, leading to apoptosis. Proteomic analysis supports the potential involvement of PC expression in the aberrant cell cycle and apoptosis, and identifies candidate proteins responsible for the PC-mediated cell cycle arrest and apoptosis in breast cancer cells.General significanceOur results highlight the possibility of the use of PC as an anti-cancer drug target.     

A Single Nucleotide Polymorphism of Chicken Acetyl-CoA Carboxylase A Gene Associated with Fatness Traits

Acetyl-CoA carboxylase α (ACCα) is a major rate-limiting enzyme in the biogenesis of long-chain fatty acids. It can catalyze the carboxylation of acetyl-CoA to form malonyl-CoA that plays a key role in the regulation of fatty acid metabolism. The objective of the present study was to investigate the associations of ACCα gene polymorphisms with chicken growth and body composition traits. The Northeast Agricultural University broiler lines divergently selected for abdominal fat content and the Northeast Agricultural University F₂ Resource Population were used in the current study. Body weight and body composition traits were measured in the aforementioned two populations. A synonymous mutation was detected in the exon 19 region of ACCα gene, then polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to genotype all the individuals derived from the aforementioned populations. Association analysis revealed that the polymorphism was associated with abdominal fat weight and percentage of abdominal fat in the two populations. The results suggested that ACCα gene could be a candidate locus or linked to a major gene that affects abdominal fat content in the chicken.     

In silico studies of urease inhibitors to explore ligand-enzyme interactions

In continuation of our previous study on the urease inhibition by a number of chalcones, 2,3-dihydro-1,5-benzothiazepines and 2,3,4,5-tetrahydro-1,5-benzothiazepines, FlexX docking has been exploited to get a deeper insight into the mechanism of their inhibitory action. A comparison of the IC₅₀ values of the active compounds reveals that, of the three classes of compounds studied, 2,3-dihydro-1,5-benzothiazepines were the most potent urease inhibitors. An in silico examination of these compounds showed that the activity is related to the interaction of ligand with the nickel metallocentre, its interaction with two amino acid residues, Asp224 and Cys322, in addition to the orientation of rings A and B in the catalytic core of the enzyme. The most active compound 2,3-dihydro-1,5-benzothiazepine (4) anchor tightly through a network of interactions with Ni701 and Ni702. This includes a number of hydrogen bonds and hydrophobic contacts with the amino acid residues in its vicinity. For their reduced analogs, the difference in the activity of different diastereomers has been observed to be configuration-dependent. This may be ascribed mainly to the difference in the orientation of ring B of the two stereoisomers and the extent of their interaction with Asp224 and Cys322 present in the catalytic core of the enzyme.     

Synthesis of D‐Altritol Nucleosides with a 3′‐O‐Tert‐Butyldimethylsilyl Protecting Group

Four D‐altritol nucleosides with a 3′‐O‐tert‐butyldimethylsilyl protecting group are synthesized (base moieties are adenine, guanine, thymine and 5‐methylcytosine). The nucleosides are obtained by ring opening reaction of 1,5:2,3‐dianhydro‐4,6‐O‐benzylidene‐D‐allitol. Optimal reaction circumstances (NaH, LiH, DBU, phase transfer, microwave irridation) for the introduction of the heterocycles are base‐specific. For the introduction of the 3′‐O‐silyl protecting group, long reaction times and several equivalents of tert‐butyldimethylsilyl chloride are needed.     

Synthesis and DNA Binding Properties of a New Benzo[f]Imidazo[1,5b]-Isoquinoline-Butan-1,2-Diol Derivative

A benzo[f]imidazo[1,5b]-isoquinoline derivative 4 with a 1,2-butandiol linker was prepared by reaction of a trimethylsilylated 5-naphthylidenehydantoin 3 with a 2,3-dideoxy-D-glycero-pentafuranoside 2 in 22% yield. After deprotection, the resulting compound 5 was converted to a DMT protected phosphoramidite building block 7 for standard DNA synthesis. DNA/DNA, DNA/RNA duplexes with 5 inserted as bulges were destabilized, except when the new amidite was used for the synthesis of a zipping duplex.     

α-MSH signalling via melanocortin 5 receptor promotes lipolysis and impairs re-esterification in adipocytes

The melanocortin system has a clear effect on the mobilisation of stored lipids in adipocytes. The aim of the current study was to investigate the role of melanocortin 5 receptor (MC5R) on α-melanocyte-stimulating hormone (α-MSH)-induced lipolysis in 3T3-L1 adipocytes. To this end, MC5R expression was decreased by small interfering RNA (siRNA), which significantly impaired the α-MSH stimulation of lipolysis, as determined by glycerol and nonesterified fatty-acid (NEFA) quantification. The functional role of α-MSH/MC5R on triglyceride (TG) hydrolysis was mediated by hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), perilipin 1 (PLIN1) and acetyl-CoA carboxylase (ACC). Immunofluorescence microscopy revealed that phosphorylated HSL clearly surrounded lipid droplets in α-MSH-stimulated adipocytes, whereas PLIN1 left the immediate periphery of lipids. These observations were lost when the expression of MC5R was suppressed.     

THE USE OF SMALL FARM DAMS IN THE TRANSKEI AND CISKEI FOR THE PRODUCTION OF TABLE-FISH USING AISCHGRUND CARP

SUMMARY

Small stockwatering dams are usually ignored in the Transkei and Ciskei in fishfarming ventures. This paper reports on experimental results in fish production studies, using Aischgrund carp, which were obtained in the Ciskei and which indicate that economically worthwhile fish production can be achieved in such dams.