Identification and Expression Analysis of the Phosphoenolpyruvate Carboxylase Gene Family in Peanut (Arachis hypogaea L.)

Phosphoenolpyruvate carboxylase （PEPC） is widely distributed in plants and bacteria, and catalyzes the carboxylation of phosphoenolpyruvate to form oxaloacetate and inorganic phosphate. To investigate the molecular mechanisms of the regulation and control of peanut oil, with the degenerated primers and RACE-PCR approach, five PEPC genes were cloned from peanut, and designated as AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4, and AhPEPC5, respectively. The structure and phylogenetic analysis of PEPC protein indicated that AhPEPC1-4 genes encoded a typical plant-type PEPC-enzyme, and AhPEPC5 a bacterial-type. By real-time quantitative RT-PCR approach the expression pattern of each gene was detected in various tissues of normal and high oil-content peanut varieties. It was found that there was a lower expression level of AhPEPCs genes except for the AhPEPC2 in high-oil peanut than normal-oil peanut line. The results provide some fundamental information for the further investigation of plant PEPC proteins and their role in regulation of oil-content in peanut seeds.

Functional Analysis of the Phosphoenolpyruvate Carboxylase on the Lipid Accumulation of Peanut(Arachis hypogaea L.) Seeds

Phosphoenolpyruvate carboxylase（PEPC;EC 4.1.1.31） catalyses phosphoenolpyruvate（PEP） to yield oxaloacetate,which is involved in protein biosynthesis.Pyruvate kinase（PK;EC 2.7.1.40） catalyzes PEP to yield pyruvate,which is involved in fatty acid synthesis.In this study,five PEPC genes（AhPEPC1,AhPEPC2,AhPEPC3,AhPEPC4,and AhPEPC5） from peanut have been cloned.Using a quantitative real-time RT-PCR approach,the expression pattern of each gene was monitored during the seed development of four peanut varieties（E11,Hebeigaoyou,Naihan 1,and Huayu 26）.It was found that these five genes shared similar expression behaviors over the developmental stages of E11 with high expression levels at 30 and 40 d after pegging（DAP）;whereas these five genes showed irregular expression patterns during the seed development of Hebeigaoyou.In Naihan 1 and Huayu 26,the expression levels of the five genes remained relatively high in the first stage.The PEPC activity was monitored during the seed development of four peanut varieties and seed oil content was also characterized during whole period of seed development.The PEPC activity followed the oil accumulation pattern during the early stages of development but they showed a significantly negative correlation thereafter.These results suggested that PEPC may play an important role in lipid accumulation during the seed development of four peanut varieties tested.

Acetyl-CoA carboxylase inhibitors in non-alcoholic steatohepatitis: Is there a benefit?

De novo lipogenesis(DNL)plays an important role in the pathogenesis of hepatic steatosis and also appears to be implicated in hepatic inflammation and fibrosis.Accordingly,the inhibition of acetyl-CoA carboxylase,which catalyzes the ratelimiting step of DNL,might represent a useful approach in the management of patients with nonalcoholic fatty liver disease(NAFLD).Animal studies and preliminary data in patients with NAFLD consistently showed an improvement in steatosis with the use of these agents.However,effects on fibrosis were variable and an increase in plasma triglyceride levels was observed.Therefore,more longterm studies are needed to clarify the role of these agents in NAFLD and to determine their risk/benefit profile.

Photosynthetic Characteristics and Heterosis in Transgenic Hybrid Rice with Maize Phosphoenolpyruvate Carboxylase (pepc) Gene

Three F3 hybrids derived from the sterile rice lines Gang 46A, 776A and 2480A and the improved restorer line Shuhui 881 containing maize phosphoenolpyruvate carboxylase （pepc） gene were used to analyze the effect of pepc gene on the heterosis and photosynthetic characteristics, while the F3 obtained by crossing Shuhui 881 with the above three sterile lines served as controls. The dynamics of photosynthetic characteristics in leaves of three F1 with pepc gene and their controls were determined at the initial-tillering, maxium-tillering, elongation, initial-heading, heading, maturity stages, and other different times after flag leaf fully expanded. The PEPCase activities of the three F1 with pepc gene increased significantly as compared with control plants during the whole developmental stages. Moreover, the net photosynthesis rate （Pn） also increased to certain extent. The data showed that PEPCase activity was significantly correlated to Pn with a correlation coefficient of 0.6081. The photosynthetic indexes of the three F1 with pepc gene were obviously superior to respective controls in apparent quantum efficiency, light compensation point and carboxylation efficiency, while the CO2 compensation point was lower than that of corresponding control. The Pn of the three F1 with pepc gene at light saturation point and CO2 saturation point was also higher than that of control plants. in addition, the three F1 with pepc gene had an average increase of 37.10% in grain yields per plant in comparison with control plants. The results indicated that the photosynthetic characteristics of hybrid rice containing pepc gene had been improved to some extent due to the introduction of pepc gene.

Genetic Mutation of Vitamin K-dependent Gamma-glutamyl Carboxylase Domain in Patients with Calcium Oxalate Urolithiasis

To investigate the exon mutation of vitamin K-dependent gamma-glutamyl carboxylase （GGCX or VKDC） in patients with calcium oxalate urolithasis, renal cortex and peripheral blood samples were obtained from severe hydronephrosis patients （with or without calculi）, and renal tumor patients undergoing nephrectomy. GGCX mutations in all 15 exons were examined in 44 patients with calcium oxalate urolithiasis （COU） by polymerase chain reaction （PCR） and denatured high pressure liquid chromatography （DHPLC）, and confirmed by sequencing. Mutation was not found in all COU samples compared to the controls. These data demonstrated that functional GGCX mutations in all 15 exons do not occur in most COU patients. It was suggested that there may be no significant association between the low activity and mutation of GGCX in COU.

Cloning, Expression and Purification of Wheat Acetyl-CoA Carboxylases CT Domain in E. coil

The entire gene of carboxyltransferase（CT） domain of acetyl-CoA carboxylase（ACCase） from Chinese Spring wheat（CSW） plastid was cloned firstly, and the 2.3 kb gene was inserted into PET28a^＋ vector and expressed in E. coil in a soluble state. The （His）6 fusion protein was identified by SDS-PAGE and Western blot. The recombinant protein was purified by affinity chromatography, and the calculated molecular mass（Mr） was 88000. The results of the sequence analysis indicate that the cloned gene（GeneBank accession No. EU124675） was a supplement and revision of the reported ACCase CT partial cDNA from Chinese Spring wheat plastid. The recombinant protein will be significant for us to investigate the recognizing mechanism between ACCase and herbicides, and further to screen new herbicides.

Concomitant Increases of the Developing Seed Phosph<i>oenol</i>pyruvate Carboxylase Ac-tivity and the Seed Protein Content of Field-Grown Wheat with Nitrogen Supply

Wheat seed storage protein is of great importance for human food. To increase the contents of storage proteins effectively, nitrogen fertilizer at flowering stages is commonly applied. In our previous study, rice phosphoenolpyruvate carboxylase (PEPCase) activity in developing seeds was observed in response to nitrogen application at a flowering stage and was positively correlated to the response of the protein content in seeds of six cultivars. This observation might indicate that the seeds have a biological system for accepting nitrogen in seeds by using PEPCase. To test whether this physiological event occurs in wheat, we examined the PEPCase activity and protein content in field-grown wheat seeds under different nitrogen supply conditions. With only basal dressing, seeds showed lower PEPCase activity and protein content (both 0.90-fold) compared to seeds without basal fertilizer. With ammonium sulfate application at 8.3 and 25 g/m2 at a flowering stage, seeds showed higher PEPCase activity (1.08- and 1.17-fold, respectively) and protein content (1.15- and 1.42-fold, respectively), depending on the nitrogen level. We investigated the relationship between PEPCase activity and protein content in the seeds among four conditions. The effect of the nitrogen supply on PEPCase activity during grain-filling stages was validated by the results of a hydroponic culture experiment. Together the results demonstrate that our hypothesis seems to apply to field-grown wheat.

Differential effect of biotin on carboxylase activity and mice skeletal muscle metabolism

In mammalian skeletal muscle there are four carboxylases involved in several biochemical processes like gluconeogenesis, tricarboxylic acid cycle anaplerosis, metabolism of fatty acids and metabolism of various amino acids. It has been shown that biotin deficiency reduces body weight at the expense of muscular mass. When necessary, the liver uses skeletal muscle protein to provide glucose and amino acids to organs in need of such compounds. In this paper we analyzed carboxylase specific activities in hind limb skeletal muscle of 3 weeks old BALB/c male mice, at 0, 1, 4, 7, and 14 days of a specific diet with different biotin concentrations. Biotin was used at 0.0, 1.8 or 98.2 mg per kg of food;and was referred to as biotin deficient, sufficient and supplemented, respectively. Water and food supply and consumption by the three groups of mice were the same. Therefore, the observed effects were directly related to biotin ingestion. The body weight of biotin supplemented mice was the same as the body weight of mice in the biotin sufficient group, while biotin deficiency caused body weight reduction after 7 days of biotin depletion. We found that the total protein concentration in the vastus lateralis muscle is associated with the biotin content in the diet. After 7 days, the muscle total protein content was lower in mice of the biotin deficient group while it was higher in the mice from the biotin supplemented group

Acetyl coenzyme A carboxylase modulates lipogenesis and sugar homeostasis in Blattella germanica

Lipid and sugar homeostasis is critical for insect development and survival.In this study,we characterized an acetyl coenzyme A carboxylase gene in Blattella germanica(BgACC)that is involved in both lipogenesis and sugar homeostasis.We found that BgACC was dominantly expressed in the fat body and integument,and was significantly upregulated after molting.Knockdown of BgACC in 5th-instar nymphs did not affect their normal molting to the next nymphal stage,but it caused a lethal phenotype during adult emergence.BgACC-RNA interference(RNAi)significantly downregulated total free fatty acid(FFA)and triacylglycerol(TAG)levels,and also caused a significant decrease of cuticular hydrocarbons(CHCs).Repression of BgACC in adult females affected the development of oocytes and resulted in sterile females,but BgACC-RNAi did not affect the reproductive ability of males.Interestingly,knockdown of BgACC also changed the expression of insulin-like peptide genes(BglLPs),which mimicked a physiological state of high sugar uptake.In addition,BgACC was upregulated when B.germanica were fed on a high sucrose diet,and repression of BgACC upregulated the expression of the glycogen synthase gene(BgGlyS).Moreover,BgACC-RNAi increased the circulating sugar levels and glycogen storage,and a longevity assay suggested that BgACC was important for the survival of B.germanica under conditions of high sucrose uptake.Our results confirm that BgACC is involved in multiple lipid biogenesis and sugar homeostasis processes,which further modulates insect reproduction and sugar tolerance.This study benefits our understanding of the crosstalk between lipid and sugar metabolism.

Promotive Effect of Low Concentrations of NaHSO3 on Photophosphorylation and Photosynthesis in Phosphoenolpyruvate Carboxylase Transgenic Rice Leaves

Spraying a 1–2 mmol/L solution of NaHSO3 on the leaves of wild-type rice (Oryza sativa L.) Kitaake (WT), phosphoenolpyruvate carboxylase (PEPC) transgenic (PC) rice and PEPC+phosphate dikinase (PPDK) transgenic rice (PC+PK), in which the germplasm was transformed with wild-type Kitaake as the gene receptor, resulted in an enhancement of the net photosynthetic rate by 23.0%, 28.8%, and 34.4%, respectively, for more than 3 d. It was also observed that NaHSO3 application caused an increase in the ATP content in leaves. Spraying PMS (a cofactor catalysing the photophosphorylation cycle) and NaHSO3 separately or together on leaves resulted in an increase in photosynthesis with all treatments. There was no additional effect on photosynthetic rate when the mixture was applied, suggesting that the mechanism by which NaHSO3 promotes photosynthesis is similar to the mechanism by which PMS acts and that both of compounds enhanced the supply of ATP. After spraying a solution of NaHSO3 on leaves, compared with the WT Kitaake rice, a greater enhancement of net photosynthetic rate was observed in PEPC transgenic (PC) and PEPC+PPDK transgenic (PC+PK) rice, with the greatest increase being observed in the latter group. Therefore ATP supply may become the limiting factor that concentrates CO2 in rice leaves transformed with an exogenous PEPC gene and exogenous PEPC+PPDK genes.

CO2 Exchange and Chlorophyll Fluorescence of Phosphoenolpyruvate Carboxylase Transgenic Rice Pollen Lines

To elucidate the photosynthetic physiological characteristics and the physiological inherited traits of rice （Oryza sativa L.） hybrids and their parents, physiological indices of photosynthetic CO2 exchange and chlorophyll fluorescence parameters were measured in leaves of the maize phosphoenolpyruvate carboxylase （PEPC） transgenic rice as the male parent, sp. japonica rice cv. 9516 as the female parent, and the stable JAAS45 pollen line. The results revealed that the PEPC gene could be stably inherited and trans- ferred from the male parent to the JAAS45 pollen line. Moreover, the JAAS45 pollen line exhibited high levels of PEPC activity, manifesting higher saturated photosynthetic rates, photosynthetic apparent quantum yield （AQY）, photochemical efficiency of photosystem II and photochemical and non-photochemical quenching, which indicated that the JAAS45 pollen line has a high tolerance to photo-inhibition/photooxidation under strong light and high temperature. Furthermore, JAAS45 was confirmed to still be a C3 plant by δ^13C carbon isotope determination and was demonstrated to have a limited photosynthetic C4 microcycle by feeding with exogenous C4 primary products, such as oxaloacetate or malate, or phosphoenolpyruvate. The present study explains the physiological inherited properties of PEPC transgenic rice and provides an expectation for the integration of traditional breeding and biological technology.

Decreased renal vitamin K-dependent γ-glutamyl carboxylase activity in calcium oxalate calculi patients

To study the activity of vitamin K-dependent γ-glutamyl carboxylase in patients with calcium oxalate (CaOx) urolithiasis compared with healthy individuals and to assess its relationship to the renal calcium oxalate urolithiasis Methods Renal parenchymas were harvested from urolithic patients and renal tumor patients undergoing nephrectomy The renal carboxylase activity was evaluated as the radioactivity of [ 14 C] labeled sodium bicarbonate in carboxylic reactions in vitro using β-liquid scintillation counting Results Significantly reduced activity of renal vitamin K-dependent γ-glutamyl carboxylase was observed in the urolithic group as compared with normal controls (P<0 01) Conclusion It suggests that the reduced carboxylase activity observed in the urolithic patients may play an important role in the course of renal calcium oxalate urolithiasis

PROPERTIES OF RuBP CARBOXYLASE/OXYGENASE IN A MUTANT TOBACCO

The aurea mutant （Su/su）of tobacco is a nuclear mutation found in Nicotiana tobacum Var. John Williams Broadleaf （Su/su）. Under greenhouse conditions and atmosphere CO2 levels, the yellow mutant tobacco （Su/su） grows much slower than the dark green wild type （su/su）. Zelitch reported that the yellow mutant has a higher photorespiratory rate and a lower net rate of photosynthesis, compared with the wild type. RuBP carboxylase/oxygenase is a key enzyme in photosynthesis and photorespiration, and the small subunit of the enzyme is nuclear-encoded. This study was designed to examine if the nuclear gene mutation influenced the structure and properties of the enzyme.

Transcritption regulation of soybean ribulose-1,5-bisphos-phate carboxylase small sub-unit gene by external factors

Ribulose-l,5-bisphosphate carboxylase small subunit gene (rbcS) is present with multi-gene family in plant genome. In Glycine max, the rbcS polypeptide (EC4.1.1.39) is encoded by a gene family containing 4-8 members. Three full-length rbcS cDNA clones were isolated and characterized from soybean seedlings, and both of their nucleotide and amino acid sequences showed high similarity. Differential accumulation of the rbcS mRNA was observed among roots, hypocotyls, cotyledons, epicotyls and leaves. The rbcS genes were up-regulated by various external factors such as salicylic acid (SA), salt stress and drought stress. The expression level of rbcS genes after being treated by 2.0 mmol/L SA and0.4% NaCl, respectively, is 2.5-3.0-fold as high as that of control sample. Moreover, soybean rbcS mRNA was accu-mulated with diurnal variation but easily influenced by light and low temperature.

IMMOBILIZATION AND SUBUNIT RECONSTITUTION OF RIBULOSE-1, 5-BISPHOSPHATE CARBOXYLASE/OXYGENASE FROM RICE

Ribulose-1, 5-bisphosphate carboxylase/oxygenase ( EC 4.1.1.39 ) ( RuBP carboxylase ) is a bifunctional enzyme which catalyzes both carboxylation and oxygenation of ribulose-1, 5-bisphosphate (RuBP). These reactions are the first step in photosynthetic carbon fixation and photorespiration respectively. Because of its key role in the formation of plant yield, many studies on the structure of active-site and catalytic mechanism of this enzyme have

A Novel Approach to Functional Analysis of the Ribulose Bisphosphate Carboxylase Small Subunit Gene by Agrobacterium-Mediated Gene Silencing

A novel approach to virus-induced post-transcriptional gene silencing for studying the function of the ribulose bisphosphate carboxylase small subunit （rbcS） gene was established and optimized using potato virus X vector and Nicotiana benthamiana as experimental material. The analysis of silencing phenomena, transcriptional level, protein expression, and pigment measurement showed that the expression of the rbcS endogenous gene was inactivated by the expression of a 500-bp homologous cDNA fragment carried in the virus vector.

Chemical synthesis of a structure gene coding for small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from tobacco

A structural gene with 385 bp, which codes for the mature small subunit (rbcS) of ribulose-1,5-bi9phosphate carboxylase/oxygenase from tobacco, has been redesigned according to the codon usage of E.coli and chemically synthesized. To facilitate the systematic investigation of structure-functional relationship of the rbcS by site-specific mutagenesis, twenty one unique restriction sites distributed throughout the synthetic gene were designed. Gene synthesis was started from chemically synthesizing sixteen oligonucleotides each with 23-66 nucleotides, and these oligonu-cleotides were annealed to form eight duplexes from which 5'- and 3'- two half molecules were formed by stepwise T4 DNA ligase reaction, and then each half molecule was cloned into plasmid pWR13, after that, two half molecules were further confirmed by DNA sequencing, finally both half molecules excised from the cloning plasmid were recombinated to form plasmid pCOTrbcS containing the whole structural gene for tobacco rbcS.

Marker-assisted Selection of ZmC_4Ppc in Rice Breeding and Yield Trait Performances of Advanced Lines

The full-length of intact Zea mays gene for phosphoenolpyruvate carboxylase gene （ZmC4Ppc） is 6 781 bp. The products of PCR for this gene were not clear with poor repeatability, resulting in that it was difficult for marker-assisted selection （MAS） both in rice and maize. For selecting the markers for MAS, sequences presented only in maize rather than in rice were identified by BLAST, and used for primer design using Primer Premier 5.0. A pair of specific primer termed MRpc （Forward： 5＇ AAGCAGGGAAGCGAGACG 3＇, Reverse： 5＇ GATTGCCGCCAGCAGTAG 3＇） was used for selection of transformed rice, and ZmC4Ppc could be highly and constitutively expressed at each tested developmental stages in the transformed rice selected by using MRpc. Thus, MRpc was used for MAS of progenies carrying ZmC4Ppc gene in rice and some restorer lines with ZmC4Ppc （e.g. FPM881） derived from ZmC4Ppc-transformed Kitaake backcrossed with a restorer line Shuhui 881 were obtained. The analyses on genetic background, PEPCase activity, net photosynthetic rate, general combining ability （GCA） and specific combining ability （SCA） of FPM881 showed that similarity of genetic background reached above 95%, the PEPCase and net photosynthetic rate were higher than those of the control, and some of the progenies carrying ZmC4Ppc gene had better GCA and SCA for grain yield per plant, number of panicles per plant, and 1000-grain weight than those of the control. This suggested that the introduction of maize ZmC4Ppc gene via MAS and its stable expression could increase grain yield of rice and would likely provide a pathway for rice varietal improvement.

Enhanced tolerance to drought in transgenic rice plants overexpressing C_4 photosynthesis enzymes

Maize-specific pyruvate orthophosphate dikinase(PPDK) was overexpressed in rice independently or in combination with the maize C4-specific phosphoenolpyruvate carboxylase(PCK). The wild-type(WT) cultivar Kitaake and transgenic plants were evaluated in independent field and tank experiments. Three soil moisture treatments,well-watered(WW), moderate drought(MD) and severe drought(SD), were imposed from 9d post-anthesis till maturity. Leaf physiological and biochemical traits, root activities,biomass, grain yield, and yield components in the untransformed WT and two transgenic rice lines(PPDK and PCK) were systematically studied. Compared with the WT, both transgenic rice lines showed increased leaf photosynthetic rate: by 20%–40% under WW, by45%–60% under MD, and by 80%–120% under SD. The transgenic plants produced 16.1%,20.2% and 20.0% higher grain yields than WT under the WW, MD and SD treatments,respectively. Under the same soil moisture treatments, activities of phosphoenolpyruvate carboxylase(PEPC) and carbonic anhydrase(CA) in transgenic plants were 3–5-fold higher than those in WT plants. Compared with ribulose-1,5-bisphosphate carboxylase, activities of PEPC and CA were less reduced under both MD and SD treatments. The transgenic plants also showed higher leaf water content, stomatal conductance, transpiration efficiency, and root oxidation activity and a stronger active oxygen scavenging system than the WT under all soil moisture treatments, especially MD and SD. The results suggest that drought tolerance is greatly enhanced in transgenic rice plants overexpressing C4photosynthesis enzymes. This study was performed under natural conditions and normal planting density to evaluate yield advantages on a field basis. It may open a new avenue to droughttolerance breeding via overexpression of C4enzymes in rice.

Metabolic pathways as possible therapeutic targets for progressive multiple sclerosis

Unlike relapsing remitting multiple sclerosis, there are very few therapeutic options for patients with progressive forms of multiple sclerosis. While immune mechanisms are key participants in the pathogenesis of relapsing remitting multiple sclerosis, the mechanisms underlying the development of progressive multiple sclerosis are less well understood. Putative mechanisms behind progressive multiple sclerosis have been put forth： insufficient energy production via mitochondrial dysfunction, activated microglia, iron accumulation, oxidative stress, activated astrocytes, Wallerian degeneration, apoptosis, etc. Furthermore, repair processes such as remyelination are incomplete. Experimental therapies that strive to improve metabolism within neurons and glia, e.g., oligodendrocytes, could act to counter inadequate energy supplies and/or support remyelination. Most experimental approaches have been examined as standalone interventions; however, it is apparent that the biochemical steps being targeted are part of larger pathways, which are further intertwined with other metabolic pathways. Thus, the potential benefits of a tested intervention, or of an established therapy, e.g., ocrelizumab, could be undermined by constraints on upstream and/or downstream steps. If correct, then this argues for a more comprehensive, multifaceted approach to therapy. Here we review experimental approaches to support neuronal and glial metabolism, and/or promote remyelination, which may have potential to lessen or delay progressive multiple sclerosis.