The Roles of Cullins E3 Ubiquitin Ligases in the Lipid Biosynthesis of the Green Microalgae Chlamydomonas reinhardtii

Microalgae-based biodiesel production has many advantages over crude oil extraction and refinement, thus attracting more and more concern. Protein ubiquitination is a crucial mechanism in eukaryotes to regulate physiological responses and cell development, which is highly related to algal biodiesel production. Cullins as the molecular base of cullin-RING E3 ubiquitin ligases (CRLs), which are the largest known class of ubiquitin ligases, control the life activities of eukaryotic cells. Here, three cullins (CrCULs) in the green microalgae Chlamydomonas reinhardtii were identified and characterized. To investigate the roles of CrCULs in lipid metabolism, the gene expression profiles of CrCULs under nutrition starvation were examined. Except for down-regulation under nitrogen starvation, the CrCUL3 gene was induced by sulfur and iron starvation. CrCUL2 seemed insensitive to nitrogen and sulfur starvation because it only had changes after treatment for eight days. CrCUL4 exhibited an expression peak after nitrogen starvation for two days but this declined with time. All CrCULs expressions significantly increased under iron deficiency at two and four days but decreased thereafter. The silencing of CrCUL2 and CrCUL4 expression using RNAi (RNA interference) resulted in biomass decline and lipids increase but an increase of 20% and 28% in lipid content after growth for 10 days, respectively. In CrCUL2 and CrCUL4 RNAi lines, the content of fatty acids, especially C16:0 and C18:0, notably increased as well. However, the lipid content and fatty acids of the CrCUL3 RNAi strain slightly changed. Moreover, the subcellular localization of CrCUL4 showed a nuclear distribution pattern. These results suggest CrCUL2 and CrCUL4 are regulators for lipid accumulation in C. reinhardtii. This study may offer an important complement of lipid biosynthesis in microalgae.     

Probing the compatibility of type II ketosynthase-carrier protein partners

Drug discovery often begins with the screening of large compound libraries to identify lead compounds. Recently, the enzymes that are involved in the biosynthesis of natural products have been investigated for their potential to generate new, diverse compound libraries. There have been several approaches toward this end, including altering the substrate specificities of the enzymes involved in natural product biosynthesis and engineering functional communication between enzymes from different biosynthetic pathways. While there exist assays to assess the substrate specificity of enzymes involved in these pathways, there is no simple method for determining whether enzymes from different synthases will function cooperatively to generate the desired product(s). Herein we report a method that provides insight into both substrate specificity and compatibility of protein-protein interactions between the acyl carrier protein (ACP) and ketosynthase (KS) domains involved in fatty acid and polyketide biosynthesis. Our technique uses a one-pot chemoenzymatic method to generate post-translationally modified ACPs that are capable of covalently interacting with KS domains from different biosynthetic systems. The extent of interaction between ACPs and KSs from different systems is easily detected and quantified by a gel-based method. Our results are consistent with previous studies of substrate specificity and ACP-KS binding interactions and provide new insight into unnatural substrate and protein interactions.     

Glyceryl-S-acyl carrier protein as an intermediate in the biosynthesis of tetronate antibiotics

The biosynthetic pathway to the unusual tetronate ring of certain polyketide natural products, including the antibiotics abyssomicin and tetronomycin (TMN) and the antitumour compound chlorothricin (CHL), is presently unknown. The gene clusters governing chlorothricin and tetronomycin biosynthesis both contain a gene encoding an atypical member of the FkbH family of enzymes, which has previously been shown to synthesise glyceryl-S-acyl carrier protein (ACP) as the first step in production of unusual extender units for modular polyketide biosynthesis. We show here that purified recombinant FkbH-like protein, Tmn16, from the TMN gene cluster catalyses the efficient transfer of a glyceryl moiety from D-1,3-bisphosphoglycerate (1,3-BPG) to either of the dedicated ACPs, Tmn7a and ChlD2, to form glyceryl-S-ACP, which directly implicates this compound as an intermediate in tetronate biosynthesis as well. Neither Tmn16 nor Tmn7a produced glyceryl-S-ACP when incubated, respectively, with analogous ACP and FkbH-like proteins from a known extender-unit pathway; this indicates a highly selective channelling of glycolytic metabolites into tetronate biosynthesis.     

Effect of nitrogen and zinc fertilization and plant growth retardants on cottonseed, protein, oil yields, and oil properties

The increase in the population in Egypt makes it imperative to explore promising approaches to increase food supply, including protein and oil, to meet the needs of the Egyptian people. Cotton is the principal crop of Egyptian agriculture. It is grown mainly for its fiber, but cottonseed products are also of economic importance. Cottonseed is presently the main source of edible oil and meal for livestock in Egypt. Field experiments were conducted in two successive seasons at the Agricultural Research Center (Giza, Egypt) on cotton (Gossypium barbadense L. cv. Giza 75) to determine the effect of nitrogen (N) fertilizer rate (107 and 161 kg of N/ha applied as ammonium nitrate containing 33.5% N in two equal doses at 6 and 8 wk after sowing), together with foliar applications of plant growth retardants (mepiquat chloride “Pix”, chloromequat chloride “Cycocel”, and daminozide “Alar”, each applied once at 288 g active ingredient/ha, after 75 d from sowing) and zinc (Zn) (applied in chelated form after 80 and 95 d from sowing at 48 g of Zn/ha) on seed, protein and oil yields and oil properties of cotton. The higher N-rate, as well as the application of all growth retardants and Zn, resulted in an increase in cottonseed yield, seed protein content, oil and protein yields/ha, seed oil refractive index, unsaponifiable matter, and total unsaturated fatty acids (oleic and linoleic). These treatments tended to decrease oil acid value, saponification value, and total saturated fatty acids. The seed oil content tended to decrease as N-rate increased and increased with the application of all growth retardants and Zn. There were some differences between Pix, Cycocel, and Alar regarding their effects on the studied characters. The highest increase in seed, oil, and protein yields/ha was found with Pix, followed by Cycocel. The Cycocel treatment gave the lowest total saturated fatty acids oil content, followed by Alar.     

A need for speed: genetic encoding of rapid cycloaddition chemistries for protein labelling in living cells

Rapid reactions: Several reactants for strain-promoted cycloaddition reactions have been genetically encoded as the side chains of noncanonical amino acids. This results in decisive improvements for the fluorescent labelling of intracellular proteins such as quantitative turnover, completion of labelling reactions within minutes, fluorogenic effects and even partial orthogonality for multicolour labelling.     

A study of simulated annealing protocols for use with molecular dynamics in protein structure prediction

The method of simulated annealing can be of use in protein structureprediction by homology modelling where side chain conformationsmust be predicted. In this study an attempt has been made tooptimize a molecular dynamics method for this purpose. Heatingand cooling protocols to maximize the accuracy of the predictionshave been developed. The optimized protocol involves coolingfrom 3000 to 0 K over 20 ps while simultaneously introducingthe non-bonded energy term. The use of a 'soft' non-bonded interactionenergy term in place of a standard 6–12 potential is foundto be important. The reliability of the predictions has beenanalysed in terms of the environment of the residues (solventaccessibility) and the degree of uncertainty in the structure(number of unknown torsion angles). Depending on these factorsthe percentage of unknown side chain torsion angles that arecorrectly predicted within 30° ranges from –50 to75%. Potential problems and limitations of the method are discussed.     

Plackett-burman design for determining the preference of Rhizomucor miehei lipase for FA in acidolysis reactions with coconut oil

The preference of lipase (EC 3.1.1.3) from Rhizomucor miehei in the incorporation of 11 FA, ranging from C10∶0 to C22∶6, into coconut oil TAG during acidolysis was studied by applying the Plackett-Burman experimental design. Enzymatic acidolysis reactions were carried out in hexane at 37°C for 48 h with coconut oil (0.1 M) and a mixture of 11 FA at a TAG to FA molar ratio of 1∶1. Lipase was used at the 5 wt% level. The incorporation of FA into coconut oil TAG was determined by GC. The lipase showed preference for long-chain saturated FA for incorporation into coconut oil TAG. The FA with 18 carbon atoms showed a high incorporation rate (18∶1>18∶1>18∶3). The lipase showed the least preference for the incorporation of 12∶0, which occurs in maximal concentration (46%), whereas the most preferred FA, 18∶0, occurs at a very low concentration (<2%) in coconut oil. The overall preference of lipase for the incorporation of different FA into coconut oil TAG was 18∶0>18∶2, 22∶0>18∶1, 18∶3, 14∶0, 20∶4, 22∶6>16∶0>12∶0≫10∶0.     

Non‐esterified fatty acids in blood cell membranes from patients with multiple sclerosis

The literature on non‐esterified fatty acid (NEFA) concentrations in blood cell membranes from patients with multiple sclerosis (MS) is scarce and reports on concentrations in brain tissue from these patients are inconsistent. NEFAs are needed for several biological functions, for example, as precursors for inflammatory eicosanoid synthesis. The objective of this study was therefore to compare NEFA concentrations in blood cell membranes from patients with that of healthy control subjects, and to correlate possible changes with disease outcome. NEFA C18:2n‐6 (9,12‐octadecadienoic acid) was decreased in peripheral blood mononuclear cell membranes from patients, median (quartile range): patients: 0.05 (0.02) and controls: 0.07 (0.14) µg/mg protein, p = 0.007. C18:2n‐6 also showed a weaker relationship with other fatty acids: with C16:0: patients: R = 0.40, p = 0.04; controls: R = 0.82, p = 0.000001. Saturated and MUFA showed positive correlations with the Bowel and bladder Functional System Scores (FSS). In contrast, in red blood cell membranes C18:2n‐6 and C22:0 (docosanoic acid) showed inverse correlations with the Sensory and Brainstem FSS. The decrease in NEFA C18:2n‐6 resulted in metabolic abnormalities between itself and saturated and monounsaturated NEFAs. Altered fatty acid composition in immune cell membranes would influence immune cell functions, and could possibly have contributed to the positive correlations between these fatty acids and disease outcome. Practical applications: Multiple sclerosis (MS) is a disease which presents with inflammation of the central nervous system. The cause of the disease is unknown and treatments such as anti‐inflammatory, immunosuppressive medications, and fatty acids supplements are for the alleviation of symptoms only. The results from this study however, showed an altered relationship between polyunsaturated and saturated as well as monounsaturated non‐esterified fatty acids in immune cells, which could have contributed to the inflammatory/infectious condition in these patients. The results from this study and further studies could possibly result in formulation of fatty acid supplements at correct doses or ratios for MS patients.     

Separation of EPA and DHA in fish oil by lipase-catalyzed esterification with glycerol

The objective of this study was to investigate the use of lipases as catalysts for separating EPA and DHA in fish oil by kinetic resolution based on their FA selectivity. Esterification of FFA from various types of fish oils with glycerol by immobilized Rhizomucor miehei lipase under water-deficient, solvent-free conditions resulted in a highly efficient separation of EPA and DHA. Reactions were conducted at 40°C with a 10% dosage of the lipase preparation under vacuum to remove the coproduced water, thus rapidly shifting the reaction toward the products. The bulk of the FA, together with EPA, were converted into acylglycerols, whereas DHA remained in the residual FFA. As an example, when FFA from tuna oil comprising 5% EPA and 25% DHA were esterified with glycerol, 90% conversion into acylglycerols was obtained after 48 h. The residual FFA contained 78% DHA and only 3% EPA, in 79% DHA recovery. EPA recovery in the acylglycerol fraction was 91%. The type of fish oil and extent of conversion were highly important parameters in controlling the degree of concentration.     

Expression of liver‐type fatty acid‐binding protein in murine lung and its release into serum upon challenge of lung with lipopolysaccharide

Fatty acid‐binding proteins (FABP) in alveolar type II (TII) cells are required for surfactant synthesis and regulation. Beyond expression of heart‐type (H‐) and epidermal‐type (E‐) FABP in TII cells from mouse lung, we present the first evidence of the expression of liver‐type (L‐) FABP, by quantitative PCR and immunofluorescent confocal laser microscopy. Further, by making use of an acute mouse lung injury model, we examine whether these lipid‐binding proteins are released into the bronchoalveolar fluid (BALF) and into the circulation upon challenge of the lung with lipopolysaccharide. Applying FABP‐specific ELISAs, we found that neither H‐ nor E‐FABP can be detected in BALF and serum above background levels, up to 24 h after insult. In contrast, L‐FABP was detected in the BALF pellet, consisting of polymorphonuclear cells and alveolar macrophages, and in serum. A significant decrease in L‐FABP levels in the BALF pellet was associated with a significant increase in serum levels 6 h post insult. As contributions of L‐FABP from other organs were excluded, this protein could be used as a marker for acute lung injury.     

重组弓形虫热休克蛋白70不同途径免疫小鼠诱导的小肠黏膜IgA抗体分泌细胞及sIgA抗体的动态变化

目的分析重组弓形虫热休克蛋白70(rTgHSP70)鼻内或皮下免疫小鼠诱导的小肠黏膜IgA抗体分泌细胞(IgA antibody-secreting cells,IgA-ASCs)与小肠冲洗液sIgA抗体的动态变化及二者的相关性,探讨其上调小肠黏膜免疫应答的作用机制。方法 BALB/c小鼠90只,随机均分3组:鼻内组(20μg rTgHSP70/只,滴鼻,免疫2次,间隔2周)、皮下组(80μg rTgHSP70/只,背部皮下注射,免疫2次,间隔2周)、对照组(不免疫)。分别于末次免疫后第1、2、3、4、5、6周,每组取小鼠5只,脱颈椎处死,免疫组化法检测十二指肠、空肠及回肠黏膜IgA-ASCs数量,ELISA法检测小肠冲洗液sIgA水平。结果 IgA-ASCs分布于小肠黏膜的固有层中,且鼻内组小肠黏膜IgA-ASCs数量明显高于皮下组和对照组(P<0.001),鼻内组小肠冲洗液sIgA水平在免后1～4周均处于上升阶段,第4周达到高峰,显著高于皮下组和对照组,差异有统计学意义(P<0.001);鼻内组和皮下组小肠冲洗液sIgA水平与十二指肠、空肠、回肠黏膜固有层IgA-ASCs的数量呈正相关(P<0.05)。结论 rTgHSP70经鼻内或皮下免疫小鼠均可诱导小肠黏膜固有层IgA-ASCs和小肠冲洗液sIgA水平的持续高表达,且二者呈正相关,鼻内免疫上调小肠黏膜免疫的作用优于皮下免疫。