Cloning of the amphibolic Calvin cycle/OPPP enzyme d-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) from spinach chloroplasts: functional and evolutionary aspects

Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme d-ribulose-5-phosphate 3-epimerase (R5P3E) (EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T₇ promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.Abbreviations R5P3E d-ribulose-5-phosphate 3-epimerase - RPI ribose-5-phosphate isomerase - TKL transketolase - PRK phosphoribulokinase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - FBP fructose-1,6-bisphophatase - FBP fructose 1,6-bisphosphate - G6PDH glucose-6-phosphate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - OPPP oxidative pentose phosphate pathway - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - FBA fructose-1,6-bisphophate aldolase - IPTG isopropyl -d-thiogalactoside - GST glutathione S-tranferase - PBS phosphate-buffered saline - TPI triosephosphate isomerase     

Molecular evolution of a portion of the mitochondrial 16S ribosomal gene region in scleractinian corals

Relationships among families and suborders of scleractinian corals are poorly understood because of difficulties 1) in making inferences about the evolution of the morphological characters used in coral taxonomy and 2) in interpreting their 240-million-year fossil record. Here we describe patterns of molecular evolution in a segment of the mitochondrial (mt) 16S ribosomal gene from taxa of 14 families of corals and the use of this gene segment in a phylogenetic analysis of relationships within the order. We show that sequences obtained from scleractinians are homologous to other metazoan 16S ribosomal sequences and fall into two distinct clades defined by size of the amplified gene product. Comparisons of sequences from the two clades demonstrate that both sets of sequences are evolving under similar evolutionary constraints: they do not differ in nucleotide composition, numbers of transition and transversion substitutions, spatial patterns of substitutions, or in rates of divergence. The characteristics and patterns observed in these sequences as well as the secondary structures, are similar to those observed in mt 16S ribosomal DNA sequences from other taxa. Phylogenetic analysis of these sequences shows that they are useful for evaluating relationships within the order. The hypothesis generated from this analysis differs from traditional hypotheses for evolutionary relationships among the Scleractinia and suggests that a reevaluation of evolutionary affinities in the order is needed. Received: 4 September 1996 / Accepted: 7 April 1997     

The generation of metabolic energy by solute transport

Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly, solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient, are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different types of secondary metabolic energy generation will be discussed.     

The relationship between amide proton chemical shifts and secondary structure in proteins

Summary The parameters for HN chemical shift calculations of proteins have been determined using data from high-resolution crystal structures of 15 proteins. Employing these chemical shift calculations for HN protons, the observed secondary structure chemical shift trends of HN protons, i.e., upfield shifts on helix formation and downfield shifts on -sheet formation, are discussed. Our calculations suggest that the main reason for the difference in NH chemical shifts in helices and sheets is not an effect from the directly hydrogen-bonded carbonyl, which gives rise to downfield shifts in both cases, but arises from an additional upfield shift predicted in helices and originating in residues i-2 and i-3. The calculations also explain the well-known relationship between amide proton shifts and hydrogen-bond lengths. In addition, the HN chemical shifts of the distorted amphipathic helices of the GCN4 leucine zipper are calculated and used to characterise the solution structure of the helices. By comparing the calculated and experimental shifts, it is shown that in general the agreement is good between residues 15 and 28. The most interesting observation is that in the N-terminal half of the zipper, although both calculated and experimental shifts show clear periodicity, they are no longer in phase. This suggests that for the N-terminal half, in the true average solution structure the period of the helix coil is longer by roughly one residue compared to the NMR structures.     

Does nitrogen fertilization have an impact on the trade-off between willow growth and defensive secondary metabolism?

The effect of nitrogen fertilization on the phytomass production, shoot length and leaf secondary phenolics in nine Salix myrsinifolia clones was investigated. Cuttings taken from 1-year-old and 2-year-old shoot parts of field cultivated clones were grown at three concentrations of nitrogen (7, 150 and 300 ppm) in a greenhouse for one growing season. The willow clones differed significantly in phytomass yield and secondary phenolics content. Nitrogen fertilization affected significantly the growth and secondary metabolism of willow clones. In most clones, the addition of nitrogen from a sub-optimum concentration (7 ppm) to an optimum concentration (150 ppm) appeared to reduce the amounts of salicortin, chlorogenic acid and unknown salicylate and increased shoot phytomass, but a supraoptimum nitrogen concentration (300 ppm) resulted in highly variable growth and secondary phenolic responses. A significantly negative correlation between leaf phytomass and amount of total phenolics at sub-optimum and optimum N-treatments indicates trade-off between growth and secondary metabolism in willow clones at these treatments. However, the leaf phytomass:total amount of phenolics ratio varied significantly among clones, and in all clones it was not significantly lower at sub-optimum N-treatment than at optimum N-treatment.     

显微红外研究巨噬细胞膜电融合后膜蛋白二级结构的变化

首次尝试了将显微傅里叶变换红外光谱术应用于研究电融合后细胞膜蛋白质二级结构的变化,发现脉冲电场作用于细胞具有穿透效应,施加电脉冲后,整个细胞的蛋白质体系能量增加,表明电泳冲对蛋白的二结构影响很大;同时,还发现用唾液酸苷酶和蛋白酶Ｐｒｏｎａｓｅ分析处理巨噬细胞膜表面后,膜上蛋白质二级结构无序化程度增加,用酶适度处理的细胞将更易发生电融合。     

牛胰多肽与CL／PC脂质体作用后二级结构的变化

用傅立叶变换红外光谱研究了ＢＰＰ与膜作用后其二级结构的变化,通过对红外光谱中酰胺Ｉ谱带进行解卷积、微分及曲线拟合等处理,结果表明,ＢＰＰ与脂膜作用后,其二级结构中α－螺旋成分增多,无规卷曲成分减少。     

The role of the secondary plant compound 2,5-dihydroxymethyl 3,4-dihydroxypyrrolidine as a feeding inhibitor for insects

The cyclic amino alditol 2,5-dihydroxymethyl 3,4-dihydroxypyrrolidine (DMDP) has recently been shown to be an inhibitor of various glucosidases. We have investigated its role as a plant protective chemical against a range of phytophagous insects by examining its effects on development, feeding behaviour and functioning of taste receptors. We have shown that it can inhibit feeding and can be toxic. The mechanisms by which these effects are achieved are discussed in the context of the molecular structure of the compound.

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Résumé Récemment a été mis en évidence le puissant pouvoir inhibiteur de différents glucosidases. Présenté par l'amino-alditol cyclique, 2,5 dihydroxymethyl 3,4 dihydroxypyrrolidine (DMDP), isolé de graines de la légumineuse Lonchocarpus sericeus. Nous avons examiné le rôle de ce composé secondaire dans la stratégie défensive de la plante contre une gamme d'insectes phytophages, à travers son action sur le développement, le comportement alimentaire et le fonctionnement des récepteurs gustatifs. Nous avons montré que DMDP peut inhiber, suivant la dose, la prise de nourriture et, qu'aprs ingestion (par Spodoptera spp), il peut agir comme une toxine. Par suite de sa structure moléculaire, DMDP peut être considéré, non seulement comme l'analogue d'un sucre, mais aussi comme un alcaloïde. Le bilan des résultats de nos expériences suggère que l'efficacité de DMDP comme phagodissuadant, si elle ne provient pas de sa toxicité, découle principalement de ses propriétés d'alcaloïde.

     

On the conservative nature of the gill filaments of sharks

Synopsis Gill filaments of one highly active and two less active shark species exhibit a conservative morphological scheme including such features as branchial canopies, marginal lamellar projections, and enlarged, discrete outer marginal lamellar channels and lateral lamellar sinuses. The specific spatial orientation of the secondary lamellae respective to one another, the gill filaments, and the interbranchial septa create what appears as one-way interfilament water channels, suggesting the presence of an efficient branchial countercurrent system. It is proposed that the fortified structure of shark gills allows many shark species to ventilate passively without having evolved gill filament modifications as apparently did some highly active teleosts. This in turn may have expedited the evolution of lamnid shark species through pre-adaptation to a swift oceanic lifestyle.