The Simultaneous Repression of CCR and CAD, Two Enzymes of the Lignin Biosynthetic Pathway, Results in Sterility and Dwarfism in Arabidopsis thaliana

Cinnamoyl CoA reductase(CCR)and cinnamyl alcohol dehydrogenase(CAD)catalyze the last steps of monolignol biosynthesis.In Arabidopsis,one CCR gene(CCR1,At1g15950)and two CAD genes(CAD C At3g19450 and CAD D At4g34230)are involved in this pathway.A triple cad c cad d ccr1 mutant,named ccc,was obtained.This mutant displays a severe dwarf phenotype and male sterility.The lignin content in ccc mature stems is reduced to 50% of the wild-type level.In addition,stem lignin structure is severely affected,as shown by the dramatic enrichment in resistant inter-unit bonds and incorporation into the polymer of monolignol precursors such as coniferaldehyde,sinapaldehyde,and ferulic acid.Male sterility is due to the lack of lignification in the anther endothecium,which causes the failure of anther dehiscence and of pollen release.The ccc hypolignified stems accumulate higher amounts of flavonol glycosides,sinapoyl malate and feruloyl malate,which suggests a redirection of the phenolic pathway.Therefore,the absence of CAD and CCR,key enzymes of the monolignol pathway,has more severe consequences on the phenotype than the individual absence of each of them.Induction of another CCR(CCR2,At1g80820)and another CAD(CAD1,At4g39330)does not compensate the absence of the main CCR and CAD activities.This lack of CCR and CAD activities not only impacts lignification,but also severely affects the development of the plants.These consequences must be carefully considered when trying to reduce the lignin content of plants in order to facilitate the lignocellulose-to-bioethanol conversion process.     

Carnitine-dependent metabolic activities in Saccharomyces cerevisiae: three carnitine acetyltransferases are essential in a carnitine-dependent strain

L-carnitine is required for the transfer of activated acyl-groups across intracellular membranes in eukaryotic organisms. In Saccharomyces cerevisiae, peroxisomal membranes are impermeable to acetyl-CoA, which is produced in the peroxisome when cells are grown on fatty acids as carbon source. In a reversible reaction catalysed by carnitine acetyltransferases (CATs), activated acetyl groups are transferred to carnitine to form acetylcarnitine which can be shuttled across membranes. Here we describe a mutant selection strategy that specifically selects for mutants affected in carnitine-dependent metabolic activities. Complementation of three of these mutants resulted in the cloning of three CAT encoding genes: CAT2, coding for the carnitine acetyltransferase associated with the peroxisomes and the mitochondria; YAT1, coding for the carnitine acetyltransferase, which is presumably associated with the outer mitochondrial membrane, and YER024w (YAT2), which encodes a third, previously unidentified carnitine acetyltransferase. The data also show that (a) L-carnitine and all three CATs are essential for growth on non-fermentable carbon sources in a strain with a disrupted CIT2 gene; (b) Yat2p contributes significantly to total CAT activity when cells are grown on ethanol; and that (c) the carnitine-dependent transfer of activated acetyl groups plays a more important role in cellular processes than previously realised.     

4‐Hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐one Synthesis by a Type III Polyketide Synthase from Rhodospirillum centenum

The purple photosynthetic bacterium Rhodospirillum centenum has a putative type III polyketide synthase gene (rpsA). Although rpsA was known to be transcribed during the formation of dormant cells, the reaction catalyzed by RpsA was unknown. Thus we examined the RpsA reaction in vitro, using various fatty acyl‐CoAs with even numbers of carbons as starter substrates. RpsA produced tetraketide pyranones as major compounds from one C_10–14 fatty acyl‐CoA unit, one malonyl‐CoA unit and two methylmalonyl‐CoA units. We identified these products as 4‐hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐ones by NMR analysis. RpsA is the first bacterial type III PKS that prefers to incorporate two molecules of methylmalonyl‐CoA as the extender substrate. In addition, in vitro reactions with ¹³C‐labeled malonyl‐CoA revealed that RpsA produced tetraketide 6‐alkyl‐4‐hydroxy‐1,5‐dimethyl‐2‐oxocyclohexa‐3,5‐diene‐1‐carboxylic acids from C_14–20 fatty acyl‐CoAs. This class of compounds is likely synthesized through aldol condensation induced by methine proton abstraction. No type III polyketide synthase that catalyzes this reaction has been reported so far. These two unusual features of RpsA extend the catalytic functions of the type III polyketide synthase family.     

Mitochondrial Glycerol-3-Phosphate Acyltransferase-Dependent Phospholipid Synthesis Modulates Phospholipid Mass and IL-2 Production in Jurkat T Cells

Changes in glycerophospholipid metabolism with age and disease can have a profound effect on immune cell activation and effector function. We previously demonstrated that glycerol‐3‐phosphate acyltransferase‐1, the first and rate limiting step in de novo glycerophospholipid synthesis, plays a role in modulating murine T cell function. The resultant phenotype is characterized by decreased IL‐2 production, increased propensity toward apoptosis, and altered membrane glycerophospholipid mass similar to that of an aged T cell. Since T cells in previous experiments were harvested from GPAT‐1^?/? mice, questions remained as to what extent the macro environment of the model influenced the observed cellular phenotype. Therefore, we generated and phenotypically characterized a mitochondrial glycerol‐3‐phosphate acyltransferase (GPAM) deficient Jurkat T cell. Furthermore, this line was used to probe possible mechanisms by which GPAT‐1/GPAM regulates T cell function. We report here that many of the key dysfunctional characteristics of murine GPAT‐1^?/? T cells are recapitulated in the GPAMKD Jurkat T cell. We found striking decreased IL‐2 production along with altered phospholipid mass and increased incidence of apoptosis. Since PtdOH is an indirect downstream product of GPAM, we attempted to rescue IL‐2 production with PtdOH supplementation; however, this addition did not return IL‐2 production to normal levels. Interestingly, we did find significantly decreased Zap‐70 phosphorylation following stimulation, suggesting that GPAM deficiency may alter membrane based stimulatory signaling. These data show for the first time that GPAM deficiency results in an inherent defect in Jurkat T cell function and glycerophospholipid composition and that this defect cannot be rescued by addition of exogenous PtdOH.     

Evaluation of hypolipidemic effects of peanut skin-derived polyphenols in rats on Western-diet

The effect of water soluble polyphenolic extract of peanut skin (PE) was investigated for its hypolipidemic properties in rats on Western diet. Seven-weeks old Wistar rats received control diet (AIN-93G), Western diet with and without a bolus of PE five times a week for 10weeks. Group which received 300mg/kg body weight showed significantly reduced body weight and epididymal fat. Plasma and liver triglyceride (TG) and cholesterol (TC) levels were significantly reduced while faecal secretion of TG and TC was greatly increased upon PE administration. Liver mRNA expression of enzymes involved in fatty acid synthesis, such as fatty acid synthase (FAS), sterol receptor element binding protein (SREBP)-1c, acetyl-CoA carboxylase (ACC1) and lipid uptake genes, such as PPARγ, were decreased, while PPARα was up-regulated by administration of PE. These data suggest that administration of PE may contribute to the improved lipid homoeostasis in rats on diets high in cholesterol and lipids.     

Molecular Dynamics of DHHC20 Acyltransferase Suggests Principles of Lipid and Protein Substrate Selectivity

Lipid modification of viral proteins with fatty acids of different lengths (S-acylation) is crucial for virus pathogenesis. The reaction is catalyzed by members of the DHHC family and proceeds in two steps: the autoacylation is followed by the acyl chain transfer onto protein substrates. The crystal structure of human DHHC20 (hDHHC20), an enzyme involved in the acylation of S-protein of SARS-CoV-2, revealed that the acyl chain may be inserted into a hydrophobic cavity formed by four transmembrane (TM) α-helices. To test this model, we used molecular dynamics of membrane-embedded hDHHC20 and its mutants either in the absence or presence of various acyl-CoAs. We found that among a range of acyl chain lengths probed only C16 adopts a conformation suitable for hDHHC20 autoacylation. This specificity is altered if the small or bulky residues at the cavity’s ceiling are exchanged, e.g., the V185G mutant obtains strong preferences for binding C18. Surprisingly, an unusual hydrophilic ridge was found in TM helix 4 of hDHHC20, and the responsive hydrophilic patch supposedly involved in association was found in the 3D model of the S-protein TM-domain trimer. Finally, the exchange of critical Thr and Ser residues in the spike led to a significant decrease in its S-acylation. Our data allow further development of peptide/lipid-based inhibitors of hDHHC20 that might impede replication of Corona- and other enveloped viruses.     

基于YOLOx残差块融合CoA模块的改进检测网络

YOLOx-Darknet53是以YOLOv3为基准增加各种技巧(trick)升级改进的检测网络,但其仍然是以Darknet53为特征提取骨干网络(backbone),因此网络的特征提取能力仍有欠缺.本文依据CoTNet中的注意力机制改进得到CoA (contextual attention)模块,并将其替代YOLOx骨干网络残差块里的3×3卷积,得到融合注意力后的新残差块,加强了骨干网络的特征提取能力,并在Pascal VOC2007数据集上进行对比实验,融合CoA模块的网络比原网络的平均精度均值AP@[.5:.95]高1.4, AP@0.5高1.4;在改进骨干网络后的YOLOx检测头前加入无参3D注意力模块,得到最终改进的检测网络,进行上述对比实验,结果表明比原网络的AP@[.5:.95]高1.6,AP@0.5高1.5.因此,改进后的网络比原网络检测更加精准,在工业应用中能达到更好的检测效果.     

A Novel Assay of DGAT Activity Based on High Temperature GC/MS of Triacylglycerol

Diacylglycerol acyltransferase (DGAT) catalyzes the final step in the acyl-CoA-dependent biosynthesis of triacylglycerol (TAG), a high-energy compound composed of three fatty acids esterified to a glycerol backbone. In vitro DGAT assays, which are usually conducted with radiolabeled substrate using microsomal fractions, have been useful in identifying compounds and genetic modifications that affect DGAT activity. Here, we describe a high-temperature gas chromatography (GC)/mass spectrometry (MS)-based method for monitoring molecular species of TAG produced by the catalytic action of microsomal DGAT. This method circumvents the need for radiolabeled or modified substrates, and only requires a simple lipid extraction prior to GC. The utility of the method is demonstrated using a recombinant type-1 Brassica napus DGAT produced in a strain of Saccharomyces cerevisae that is deficient in TAG synthesis. The GC/MS-based assay of DGAT activity was strongly correlated with the typical in vitro assay of the enzyme using [1-¹⁴C] acyl-CoA as an acyl donor. In addition to determining DGAT activity, the method is also useful for determining substrate specificity and selectivity properties of the enzyme.     

caTBUA: Context‐aware ticket‐based binding update authentication protocol for trust‐enabled mobile networks

Existing binding update (BU) authentication protocols do not consider context information, such as trust, location, and current time, when verifying a mobile node's care‐of address (CoA). Instead, the correspondent node executes its own CoA validation in spite of facing a highly trusted situation or simply bypasses the CoA validation, making it difficult to maintain a reasonable trade‐off between security and efficiency. This paper applies the context‐aware concept to the BU process and proposes a new context‐aware ticket‐based binding update authentication (caTBUA) protocol. The proposed protocol dynamically performs an appropriate CoA validation based on the context information to achieve a good balance between security and efficiency. Utilizing numerical analysis to compare the performance of the proposed protocol to that of existing authentication protocols in terms of authentication cost and authentication message transmission latency confirmed that the proposed caTBUA protocol yields a better performance than the existing BU authentication protocols. Copyright © 2010 John Wiley & Sons, Ltd.     

Dietary Hyodeoxycholic Acid Exerts Hypolipidemic Effects by Reducing Farnesoid X Receptor Antagonist Bile Acids in Mouse Enterohepatic Tissues

Mice were fed a control diet or a diet supplemented with hyodeoxycholic acid, the most abundant bile acid contained in pig bile, for 4 weeks, after which their serum and livers were collected. The contents of total fatty acids of serum and liver cholesteryl esters, and of liver triglycerides, were reduced following the administration of the hyodeoxycholic acid‐supplemented diet, which was mainly due to the reductions in the contents of monounsaturated fatty acids. Free cholesterol contents in the serum and liver were not changed by hyodeoxycholic acid administration. Hyodeoxycholic acid administration reduced the gene expression levels of sterol regulatory element binding protein 1c, acetyl‐CoA carboxylase, fatty acid synthase, and stearoyl‐CoA desaturase‐1. Hyodeoxycholic acid administration markedly changes the ratio of FXR‐antagonist/FXR‐agonist bile acids in the enterohepatic tissues of the mice (1.13 and 7.60 in hyodeoxycholic acid and control diet groups, respectively). Our findings demonstrate that hyodeoxycholic acid administration exerts the hypolipidemic effect in mice, in which downregulations of de novo lipogenesis and desaturation of saturated fatty acids are suggested to play important roles. In addition, regulation of FXR activation through the selective modification of the enterohepatic bile acid pool may be involved in the hypolipidemic effect of hyodeoxycholic acid administration.