Competition between isoprene emission and pigment synthesis during leaf development in aspen

In growing leaves, lack of isoprene synthase (IspS) is considered responsible for delayed isoprene emission, but competition for dimethylallyl diphosphate (DMADP), the substrate for both isoprene synthesis and prenyltransferase reactions in photosynthetic pigment and phytohormone synthesis, can also play a role. We used a kinetic approach based on post‐illumination isoprene decay and modelling DMADP consumption to estimate in vivo kinetic characteristics of IspS and prenyltransferase reactions, and to determine the share of DMADP use by different processes through leaf development in Populus tremula. Pigment synthesis rate was also estimated from pigment accumulation data and distribution of DMADP use from isoprene emission changes due to alendronate, a selective inhibitor of prenyltransferases. Development of photosynthetic activity and pigment synthesis occurred with the greatest rate in 1‐ to 5‐day‐old leaves when isoprene emission was absent. Isoprene emission commenced on days 5 and 6 and increased simultaneously with slowing down of pigment synthesis. In vivo Michaelis–Menten constant (K_m) values obtained were 265 nmol m^?2 (20 μm ) for DMADP‐consuming prenyltransferase reactions and 2560 nmol m^?2 (190 μm ) for IspS. Thus, despite decelerating pigment synthesis reactions in maturing leaves, isoprene emission in young leaves was limited by both IspS activity and competition for DMADP by prenyltransferase reactions.     

斯达油脂酵母中自噬与油脂积累的关联性分析

旨在证明斯达油脂酵母中自噬参与油脂积累过程。在不同油脂积累水平下,检测相关自噬基因的表达,观察自噬与油脂积累是否有潜在相关性;用自噬促进剂促进自噬后,观察酵母油脂积累水平是否有差异。结果表明,与油脂低积累水平相比,在油脂高积累条件下,斯达油脂酵母自噬水平较低;用自噬促进剂处理后,酵母油脂积累降低。在斯达油脂酵母中,自噬与油脂积累成负相关性,自噬水平的提高会使酵母中油脂积累降低。     

VPg unlinkase,the phosphodiesterase that hydrolyzes the bond between VPg and picornavirus RNA: a minimal nucleic moiety of the substrate

VPg unlinkase is an unusual eukaryotic enzyme that catalyzes hydrolysis of the phosphodiester bond between residues of the unique tyrosine of VPg (viral protein genome-linked) and the 5"-terminal uridylic acid of picornavirus RNA. Cellular targets of the VPg unlinking enzyme are yet unknown. To determine an essential nucleic part of the covalent linkage unit that is necessary for the VPg unlinkase reaction, the following derivatives of the encephalomyocarditis virus (EMCV) VPg–RNA complex were used: [¹²⁵I]Kp–pUpUpGp, [¹²⁵I]Kp–pUp, and [¹²⁵I]Kp–pU (Kp is residual peptides bound to RNA after proteinase K treatment of VPg–RNA). [¹²⁵I]K-peptides were unlinked from [¹²⁵I]Kp–pUpUpGp and [¹²⁵I]Kp–RNA with similar velocity, but [¹²⁵I]Kp–pUp was split much slower. Under the same conditions [¹²⁵I]Kp–pU was not dissociated at all. Thus, pUp is a minimal part of picornavirus RNA that is necessary for VPg unlinkase. We speculate that cellular substrates of the enzyme are phosphodiesters of oligo(poly)ribonucleotides and tyrosine or tyrosine peptides. In no case [¹²⁵I]VPg–pU, [¹²⁵I]VPg–pUp, and [¹²⁵I]VPg–pUpUpGp were hydrolyzed by VPg unlinkase, in contrast with [¹²⁵I]VPg–RNA and [¹²⁵I]VPg–pUpUpGpApApApGp. We conclude that the whole VPg, when bound to trinucleotide (but not to heptanucleotide), protects the inter-polymeric phosphodiester bond against hydrolysis of the covalent linkage unit. We speculate that VPg unlinkase might repair covalent complexes of RNA and topoisomerases and trigger degradation process of the picornavirus RNA.     

Dual Localization of Squalene Epoxidase, Erg1p, in Yeast Reflects a Relationship between the Endoplasmic Reticulum and Lipid Particles

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 × g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Δ²⁴-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.     

Heme biosynthesis in mammalian systems: evidence of a Schiff base linkage between the pyridoxal 5'-phosphate cofactor and a lysine residue in 5-aminolevulinate synthase.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in nonplant higher eukaryotes. Murine erythroid 5-aminolevulinate synthase has been purified to homogeneity from an Escherichia coli overproducing strain, and the catalytic and spectroscopic properties of this recombinant enzyme were compared with those from nonrecombinant sources (Ferreira, G.C. & Dailey, H.A., 1993, J. Biol. Chem. 268, 584-590). 5-Aminolevulinate synthase is a pyridoxal 5'-phosphate-dependent enzyme and is functional as a homodimer. The recombinant 5-aminolevulinate synthase holoenzyme was reduced with tritiated sodium borohydride and digested with trypsin. A single peptide contained the majority of the label. The tritiated peptide was isolated, and its amino acid sequence was determined; it corresponded to 15 amino acids around lysine 313, to which pyridoxal 5'-phosphate is bound. Significantly, the pyridoxyllysine peptide is conserved in all known cDNA-derived 5-aminolevulinate synthase sequences and is present in the C-terminal (catalytic) domain. Mutagenesis of the 5-aminolevulinate synthase residue, which is involved in the Schiff base linkage with pyridoxal 5'-phosphate, from lysine to alanine or histidine abolished enzyme activity in the expressed protein.     

An unusual beta-ketoacyl:acyl carrier protein synthase and acyltransferase motifs in TaK, a putative protein required for biosynthesis of the antibiotic TA in Myxococcus xanthus

The antibiotic TA of Myxococcus xanthus is produced by a type-I polyketide synthase mechanism. Previous studies have indicated that TA genes are clustered within a 36-kb region. The chemical structure of TA indicates the need for several post-modification steps, which are introduced to form the final bioactive molecule. These include three C-methylations, an O-methylation and a specific hydroxylation. In this study, we describe the genetic analysis of taK, encoding a specific polyketide beta-ketoacyl:acyl carrier protein synthase, which contains an unusual beta-ketoacyl synthase and acyltransferase motifs and is likely to be involved in antibiotic TA post-modification. Functional analysis of this beta-ketoacyl:acyl carrier protein synthase by specific gene disruption suggests that it is essential for the production of an active TA molecule.     

Structure of a mutant human purine nucleoside phosphorylase with the prodrug, 2‐fluoro‐2′‐deoxyadenosine and the cytotoxic drug, 2‐fluoroadenine

A double mutant of human purine nucleoside phosphorylase (hDM) with the amino acid mutations Glu201Gln:Asn243Asp cleaves adenosine‐based prodrugs to their corresponding cytotoxic drugs. When fused to an anti‐tumor targeting component, hDM is targeted to tumor cells, where it effectively catalyzes phosphorolysis of the prodrug, 2‐fluoro‐2′‐deoxyadenosine (F‐dAdo) to the cytotoxic drug, 2‐fluoroadenine (F‐Ade). This cytotoxicity should be restricted only to the tumor microenvironment, because the endogenously expressed wild type enzyme cannot use adenosine‐based prodrugs as substrates. To gain insight into the interaction of hDM with F‐dAdo, we have determined the crystal structures of hDM with F‐dAdo and F‐Ade. The structures reveal that despite the two mutations, the overall fold of hDM is nearly identical to the wild type enzyme. Importantly, the residues Gln201 and Asp243 introduced by the mutation form hydrogen bond contacts with F‐dAdo that result in its binding and catalysis. Comparison of substrate and product complexes suggest that the side chains of Gln201 and Asp243 as well as the purine base rotate during catalysis possibly facilitating cleavage of the glycosidic bond. The two structures suggest why hDM, unlike the wild‐type enzyme, can utilize F‐dAdo as substrate. More importantly, they provide a critical foundation for further optimization of cleavage of adenosine‐based prodrugs, such as F‐dAdo by mutants of human purine nucleoside phosphorylase.     

An efficient approach for cloning the dNDP-glucose synthase gene from actinomycetes and its application in Streptomyces spectabilis, a spectinomycin producer

Specifically designed PCR primers were applied to amplify a segment of dTDP-glucose synthase gene from six actinomycete strains. About 300-bp or 580-bp DNA fragments were obtained from all the organisms tested. By DNA sequence analysis, seven amplified fragments showed high homology with dTDP-glucose synthase genes that participate in the biosynthesis of secondary metabolites or in deoxy-sugar moieties in lipopolysaccharides. In addition, we have cloned a 45-kb region of DNA from Streptomyces spectabilis ATCC27741, a spectinomycin producer which contained the dTDP-glucose synthase and dTDP-glucose 4,6-dehydratase genes named spcD and spcE, respectively. The spcE gene was expressed in Escherichia coli and the activity was assayed in cell extracts. The enzyme showed substrate specificity only to dTDP-glucose.     

Relationship between a host plant compound,myrcene and pheromone production in the bark beetle, IPS paraconfusus

The pheromonal components, ipsenol and ipsdienol were found in increasing quantities in hindguts of only the male sex of Ips paraconfusus following exposure of both sexes to a series of increasing concentrations of myrcene vapour. Hindguts of female and male beetles contained similar quantities of myrcene and other volatile compounds associated with myrcene exposure. Unexposed beetles of both sexes did not contain detectable amounts of any volatile compound. This indicates that myrcene induces or is a precursor for sex-specific pheromone biosynthesis.     

Relationships between CO2 concentration within the leaf and photosynthesis during deacidification in a CAM plant, Kalanchoë pinnatum

The CO₂ concentration within the leaf of Kalanchoë pinnatum (Persoon) was measured during deacidification in the light. When the acidified leaf was treated with DCMU, the CO₂ concentration within the leaf was increased about 3-fold as compared to that of the non-treated leaf, concomitant with a decline in deacidification. Low light intensity during deacidification also increased CO₂ concentration within the leaf. From these experiments, it was concluded that in order for deacidification to proceed, CO₂ released from malate must be fixed continuously by photosynthesis. Otherwise, an equilibrium for malate decomposition is soon established and results in suppression of deacidification. Thus, the CO₂ concentration within the leaf seems to be one of the regulatory factors of deacidification.     

褐飞虱体内类酵母共生菌与氨基酸营养的关系

利用全纯人工饲料饲喂技术,研究了缺失不同氨基酸对高温(35℃)处理后的缺菌褐飞虱Nilaparvata lugens Stål相对生长速度、体内共生菌数量的影响,发现10种必需氨基酸对缺菌褐飞虱生长的影响明显大于10种非必需氨基酸,饲料中必需氨基酸的缺少对褐飞虱（特别是高温处理褐飞虱）体内共生菌数量有一定的刺激作用。分析了缺菌试虫体内氨基酸组成和转氨酶活性的变化规律,发现在摄取的氨基酸营养相同的条件下（用全纯饲料D-97饲养）,高温处理试虫体内蛋白质氨基酸组成无明显变化,而游离氨基酸总量明显上升,且必需氨基酸所占比例显著下降,其中组氨酸（His）、异亮氨酸（Ile）、亮氨酸（Leu）、赖氨酸（Lys）、蛋氨酸（Met）和苯丙氨酸（Phe）摩尔百分含量均显著下降,表明必需氨基酸的相对缺乏可能是体内蛋白质合成受阻的一个重要原因,推测这可能是由于试虫体内共生菌数减少致使所合成的必需氨基酸减少而引起。处理试虫体内谷氨酰胺合成酶（GS）和丙氨酸氨基转移酶（ALT）活性明显提高,天冬氨酸氨基转移酶(AST)活性显著降低,结合游离氨基酸中谷氨酰胺（Gln）显著增多,推测类酵母共生菌可能利用谷氨酰胺等为原料进行必需氨基酸的合成。     

Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan-synthase beta chain.

The isolated, 101-residue long C-terminal (so called F2) fragment of the beta chain from Escherichia coli tryptophan synthase was shown previously to fold into an ensemble of conformations that are condensed, to contain large amounts of highly dynamic secondary structures, and to behave as a good model of structured intermediates that form at the very early stages of protein folding. Here, solvent perturbations were used to investigate the forces that are involved in stabilizing the secondary structure (monitored by far-UV CD) and the condensation of the polypeptide chain (monitored by dynamic light scattering) in isolated F2. It was observed that neither the ionic strength, nor the pH (between 7 and 10), nor salts of the Hofmeister series affected the global secondary structure contents of F2, whereas some of these salts affected the collapse slightly. Addition of trifluoroethanol resulted in a large increase in both the amount of secondary structure and the Stokes radius of F2. Conversely, F2 became more condensed upon raising the temperature from 4 to 60 degrees C, whereas in this temperature range, the secondary structure undergoes significant melting. These observations lead to the conclusion that, in isolated F2, there is no coupling between the hydrophobic collapse and the secondary structure. This finding will be discussed in terms of early events in protein folding.     

Characterization of a novel nm23 gene and its potential roles in gametogenesis in the prawn Macrobrachium rosenbergii (de Man, 1879) (Crustacea: Decapoda)

Nm23 is a family of genes encoding the nucleoside diphosphate (NDP) kinase, which functions in a wide variety of biological processes, including growth, development, differentiation and tumor metastasis. In this study, a novel nm23 gene, designated as Mrnm23, was identified from the freshwater giant prawn Macrobrachium rosenbergii. The full-length cDNA was 776 bp in length, encoding for a protein of 176 amino acids with one typical NDP kinase domain that harbored all the crucial residues for nucleotide binding and enzymatic activity. Like human novel nm23-H1B, the putative protein contained a unique 21-amino-acid NH₂-terminal extension as compared to human nm23 (nm23-H1) homologs. Further, 3 extra amino acid residues prolonged the COOH-terminus. The Mrnm23 was ubiquitously expressed in all tissues examined, including androgenic gland, gill, heart, liver, muscle, ovary, and testis. In situ hybridization to gonad sections indicated that the Mrnm23 mRNA was localized in the cytoplasm of cup-base of differentiating spermatids, in the spike of the umbrella-shaped spermatozoa and in the cytoplasm of the early previtellogenic oocytes, suggesting that the Mrnm23 has potential roles in spermiogenesis and early differentiation of oocyte.     

Association between chemerin rs17173608 and vaspin rs2236242 gene polymorphisms and the metabolic syndrome,a preliminary report

Metabolic syndrome (MeS) is associated with increased risk for type 2 diabetes mellitus (T2DM) and cardiovascular disease. There is some evidence indicating that adipokines play a role in the development of MeS. The present study was aimed to investigate the impact of chemerin rs17173608 and vaspin rs2236242 gene polymorphisms with the risk of MeS in a sample of Iranian population.     

Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana

The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.     

Use of spent mushroom substrates from Agaricus subrufescens (syn. A. blazei, A. brasiliensis) and Lentinula edodes productions in the enrichment of a soil-based potting media for lettuce (Lactuca sativa) cultivation: Growth promotion and soil bioremediation

This study aimed to assess physicochemical and microbiological properties of fresh spent mushroom substrates (SMSs) – without post-crop heat treatment – from Agaricus subrufescens and Lentinula edodes production to optimize the use of these residues in the soil enrichment for lettuce growth promotion and soil remediation. Organic matter and C content of both SMSs were high. Fresh A. subrufescens SMS was a good source of N, P and K. On the other hand, L. edodes SMS presented a lower concentration of these nutrients and a high level of immaturity. Both SMSs presented high electric conductivity values (2.5–3.4 mS/cm). Microbiological analysis, based upon enumeration of culturable bacteria (thermophilic and mesophilic) and fungi, and also evolution of CO₂, showed that SMSs played higher microbial diversity than soil control. Laccase activity from A. subrufescens SMS tended to remain constant during a 2-month period, while L. edodes SMS presented low laccase activity throughout the same period. Agaricus subrufescens and L. edodes were able to grow on a PDA (Potato Dextrose Agar) media supplemented with different concentrations of atrazine (1–50 μg/ml), degraded the herbicide, attaining rates of 35% and 26%, respectively. On experiments of lettuce growth promotion using a soil-based potting media with different SMS rates, 5% and 10% (dw) rates of A. subrufescens SMS resulted in higher lettuce aerial dry weights than the rates of 25% and 40%, the chemical fertilization (NPK) and the control (soil). At 10% supplementation, lettuce aerial dry weight increased 2.2 and 1.3 times compared to the control and the NPK treatment, respectively. Protein content increased along with SMS rates. Fresh A. subrufescens SMS was an excellent supplement for lettuce growth promotion and showed potential for remediation of biocides possibly due to improved microbial diversity and enzymatic activity. Fresh L. edodes SMS was not a good fertilizer, at least under the conditions tested. However, microbiological analysis showed that promising results may be achieved when using fresh L. edodes SMS for soil remediation.     

Maternal PCMT1 gene polymorphisms and the risk of neural tube defects in a Chinese population of Lvliang high-risk area

Protein-L-isoaspartate (D-aspartate) O-methyltransferase 1 (PCMT1) gene encodes for the protein repair enzyme L-isoaspartate (D-aspartate) O-methyltransferase (PIMT), which is known to protect certain neural cells from Bax-induced apoptosis. Previous study has shown that PCMT1 polymorphisms rs4552 and rs4816 of infant are associated with spina bifida in the Californian population. The association between maternal polymorphism and neural tube defects is still uncovered. A case-control study was conducted to investigate a possible association between maternal PCMT1 and NTDs in Lvliang high-risk area of Shanxi Province in China, using a high-resolution DNA melting analysis genotyping method. We found that increased risk for anencephaly in isolated NTDs compared with the normal control group was observed for the G (vs. A) allele (p=0.034, OR=1.896, 95% CI, 1.04-3.45) and genotypes GG+GA (p=0.025, OR=2.237, 95% CI, 1.09-4.57). Although the significance was lost after multiple comparison correction, the results implied that maternal polymorphisms in PCMT1 might be a potential genetic risk factor for isolated anencephaly in this Chinese population.