Developmental regulation of cytokinin, spore germination inhibitor discadenine and related enzymes in Dictyostelium discoideum

The amounts of discadenine (spore germination inhibitor of Dictyostelium discoideum) and its precursor, N⁶-Δ²-isopentenyladenine (i⁶Ade) in cells of D. discoideum were measured at various stages of differentiation. The activities of the enzymes involved in the biosynthesis of these bases, i.e., discadenine synthetase and 5′-AMP: Δ²-isopentenylpyrophosphate Δ²-isopentenyltransferase (5′-AMP isopentenyltransferase) in the cells were also measured. During differentiation, discadenine appeared in the cells at the stage of culmination before i⁶Ade was detected. The activity of 5′-AMP: Δ²-isopentenylpyrophosphate Δ²-isopentenyltransferase in cell extracts increased after the beginning of culmination, much later than the increase in discadenine synthetase activity. The order of appearance of these bases and enzymes is apparently the reverse of that expected from the biosynthetic route. The implications of these findings are discussed.     

Purification and some properties of Δ2-isopentenylpyrophosphate:5′AMP Δ2-isopentenyltransferase from the cellular slime mold Dictyostelium discoideum

Δ²-Isopentenylpyrophosphate:5′AMP Δ²-isopentenyltransferase, which catalyzes the formation of isopentenyl-AMP from Δ²-isopentenylpyrophosphate and 5′AMP, was purified 6800-fold from the fruiting body of the cellular slime mold Dictyostelium discoideum using several separation procedures including 5′AMP^ox-redAH-Sepharose 4B affinity column chromatography. The final preparation was very unstable and lost its activity in a day. Various properties of the 1000-fold-purified enzyme preparation were examined. The molecular mass was 40,000 ± 2000 Da, as determined by Sephadex G-100 superfine gel filtration. The divalent metal ions Mn²⁺, Zn²⁺, and Mg²⁺ profoundly affected the enzymatic activity depending on their concentration, and also altered the optimum pH and temperature. Of the compounds tested, 5′AMP was the best acceptor of the isopentenyl group and, interestingly, ADP also served as a substrate, being 60–80% as effective as 5′AMP. Adenine, adenosine, and ATP were not substrates for this enzyme. Under the optimum assay conditions (pH 7.0, 1 mm Zn²⁺, and 25 °C) the K_m values for 5′AMP and Δ²-isopentenylpyrophosphate were 1.0 × 10^?7m and 2.2 × 10^?6m, respectively.     

Stereochemistry of Pyrethrosin,Cyclopyrethrosin Acetate and Isocyclopyrethrosin Acetate

The acid cyclization product from pyrethrosin has been proved to be a (1:1)-mixture of cyclopyrethrosin acetate containing a Δ³⁽⁴⁾-double bond and isocyclopyrethrosin acetate with a Δ⁴⁽¹⁵⁾-double bond through the reinvestigation. NMR and ORD studies on their derivatives led us to assign revised stereochemistry to pyrethrosin and its related compounds.     

Laboratory metabolic evolution improves acetate tolerance and growth on acetate of ethanologenic Escherichia coli under non-aerated conditions in glucose-mineral medium

In this work, Escherichia coli MG1655 was engineered to produce ethanol and evolved in a laboratory process to obtain an acetate tolerant strain called MS04 (E. coli MG1655: ΔpflB, ΔadhE, ΔfrdA, ΔxylFGH, ΔldhA, PpflB::pdc _Zm -adhB _Zm , evolved). The growth and ethanol production kinetics of strain MS04 were determined in mineral medium, mainly under non-aerated conditions, supplemented with glucose in the presence of different concentrations of sodium acetate at pH?7.0 and at different values of acid pH and a constant concentration of sodium acetate (2?g/l). Results revealed an increase in the specific growth rate, cell mass formation, and ethanol volumetric productivity at moderate concentrations of sodium acetate (2–10?g/l), in addition to a high tolerance to acetate because it was able to grow and produce a high yield of ethanol in the presence of up to 40?g/l of sodium acetate. Genomic analysis of the ΔpflB evolved strain identified that a chromosomal deletion of 27.3?kb generates the improved growth and acetate tolerance in MG1655 ΔpflB derivative strains. This deletion comprises genes related to the respiration of nitrate, repair of alkylated DNA and synthesis of the ompC gene coding for porin C, cytochromes C, thiamine, and colonic acid. Strain MS04 is advantageous for the production of ethanol from hemicellulosic hydrolysates that contain acetate.     

Neofunctionalization in an ancestral insect desaturase lineage led to rare Δ6 pheromone signals in the Chinese tussah silkworm

The Chinese tussah silkworm, Antheraea pernyi (Lepidoptera: Saturniidae) produces a rare dienoic sex pheromone composed of (E,Z)-6,11-hexadecadienal, (E,Z)-6,11-hexadecadienyl acetate and (E,Z)-4,9-tetradecadienyl acetate, and for which the biosynthetic routes are yet unresolved. By means of gland composition analyses and in vivo labeling we evidenced that pheromone biosynthesis towards the immediate dienoic gland precursor, the (E,Z)-6,11-hexadecadienoic acid, involves desaturation steps with Δ⁶ and Δ¹¹ regioselectivity. cDNA cloning of pheromone gland desaturases and heterologous expression in yeast demonstrated that the 6,11-dienoic pheromone is generated from two biosynthetic routes implicating a Δ⁶ and Δ¹¹ desaturase duo albeit with an inverted reaction order. The two desaturases first catalyze the formation of the (E)-6-hexadecenoic acid or (Z)-11-hexadecenoic acid, key mono-unsaturated biosynthetic intermediates. Subsequently, each enzyme is able to produce the (E,Z)-6,11-hexadecadienoic acid by accommodating its non-respective mono-unsaturated product. Besides elucidating an unusually flexible pheromone biosynthetic pathway, our data provide the first identification of a biosynthetic Δ⁶ desaturase involved in insect mate communication. The occurrence of this novel Δ⁶ desaturase function is consistent with an evolutionary scenario involving neo-functionalization of an ancestral desaturase belonging to a gene lineage different from the Δ¹¹ desaturases commonly involved in moth pheromone biosynthesis.     

The in vivo metabolism of progestins: IV. the metabolic clearance rate and plasma binding of 6α-methylpregn-4-ene-3,20-dione in women

[³H]6α-methylprogesterone (6MP) was synthesized by selective catalytic tritiation of the Δ¹-olefinic bond of 6α-methylpregna-1,4-diene-3,20-dione. The metabolic clearance rate of 6MP (MCR⁶MP) was determined in 6 women by the single injection technique. The plasma MCR^6MP was 4047 ± 298 L/day (59 ± 15 L/day/kg) which was higher than the MCR of progesterone and medroxyprogesterone acetate (6α-methyl-17α-hydroxy-pregna-4-ene-3,20-dione acetate). The high clearance was not due to binding or metabolism of 6MP by red cells. Although 6MP was bound to CBG with a lower affinity than progesterone, this could not entirely explain the high MCR^6MP. When considered with the reports of progesterone and medroxyprogesterone acetate clearance, the present studies suggest that the 6α-substitution of progesterone leads to an increased rate of steroid metabolism in women.     

Isomerization and Racemization of Menthols

Menthols were converted to Δ³-menthone enol acetate (VII) via menthones having only one asymmetric carbon atom. It was shown that the optical rotation of menthone enol acetate was proportional to the optical purity of starting menthols. Optical purity of original menthol could, therefore, be determined by optical rotation of menthone enol acetate derived from.     

A role for EIIANtr in controlling fluxes in the central metabolism of E. coli K12

To investigate a possible role of the nitrogen-PTS (PTS^Ntr) in controlling carbon metabolism, we determined the growth of Escherichia coli LJ110 and of isogenic derivatives, mutated in components of the PTS^Ntr, on different carbon sources. The PTS^Ntr is a set of proteins homologous to the PEP-dependent phosphotransferase system (C-PTS) that transfers a phosphate group from PEP over EI^Ntr (encoded by ptsP) and NPr (encoded by ptsO) to EIIA^Ntr (encoded by ptsN). Strains deleted in ptsN were characterized by a high acetate production coupled to slow growth on glycolytic substrates. The ΔptsP and the ΔptsO strain showed the same behavior as the parent strain. As the phosphorylation level of EIIA^Ntr in these mutants differed significantly from that of the parent strain, phosphorylation of EIIA^Ntr obviously is not important for its function. During growth in minimal medium with defined carbon sources, EIIA^Ntr was always completely phosphorylated in LJ110. Significant amounts of dephosphorylated EIIA^Ntr were only visible in strains lacking EI^Ntr or NPr. mRNA expression studies on glucose revealed a downregulation of genes encoding TCA cycle enzymes when EIIA^Ntr was absent. ¹³C-flux analyses confirmed higher fluxes towards acetate and lower fluxes in the TCA cycle in the ptsN mutants but additionally hinted to a slightly but significantly increased flux through the pyruvate dehydrogenase complex (PDH). During growth on succinate the ΔptsN strain accumulated mutations in rpoS, while no rpoS mutants were observed for the ΔptsN-O strain. This hints to an additional function of NPr during growth with succinate.     

Genetic Analysis of Carbon Isotope Discrimination and its Relation to Yield in a Wheat Doubled Haploid Population

Carbon isotope discrimination (Δ¹³C) is considered a useful indicator for indirect selection of grain yield (GY) in cereals. Therefore, it is important to evaluate the genetic variation in Δ¹³C and its relationship with GY. A doubled haploid (DH) population derived from a cross of two common wheat varieties, Hanxuan 10 (H10) and Lumai 14 (L14), was phenotyped for Δ¹³C in the flag leaf, GY and yield associated traits in two trials contrasted by water availability, specifically, rain‐fed and irrigated. Quantitative trait loci (QTLs) were identified by single locus and two locus QTL analyses. QTLs for Δ¹³C were located on chromosomes 1A, 2B, 3B, 5A, 7A and 7B, and QTLs for other traits on all chromosomes except 1A, 4D, 5A, 5B and 6D. The population selected for high Δ¹³C had an increased frequency of QTL for high Δ¹³C, GY and number of spikes per plant (NSP) when grown under rain‐fed conditions and only for high Δ¹³C and NSP when grown under irrigated conditions, which was consistent with agronomic performance of the corresponding trait values in the high Δ¹³C progeny; that is, significantly greater than that in the low Δ¹³C. Therefore, selection for Δ¹³C was beneficial in increasing grain yield in rain‐fed environments.     

The synthesis of 2α,3α-isopropylidenedioxy-6,6-ethylenedioxy-5α-androst-15-en-17-one and its 2β,3β-isomer

Androstane and Δ¹⁵-androstane analogues of brassinosteroids were synthesized from dehydroepiandrosterone. The key stage, hydroxylation of 17β-acetoxyandrost-2-en-6-one double bond with OsO₄, yielded the corresponding 2α,3α-and 2β,3β-diols. The target 2α,3α-isopropylidenedioxy-6,6-ethylenedioxy-5α-androst-15-en-17-one and its 2β,3β-isomer were obtained by dehydrosilylation of the corresponding silylenol ethers with palladium acetate.     

Inhibition of hydrocarbon biosynthesis in the housefly by 2-Octadecynoate

The elongation of [9,10-³H]oleoyl-CoA with malonyl-CoA to form 20, 22, and 24 carbon monounsaturated fatty acids was demonstrated in housefly microsomes by radio-GLC. These elongation reactions, which have been postulated to be involved in hydrocarbon biosynthesis, have not been previously demonstrated in insects. 2-Octadecynoate (18:1 Δ²⁼) inhibited the in vivo incorporation of [1-¹⁴C]acetate into both fatty acids and hydrocarbons in a dose-dependent manner. At doses of 10 μg per female housefly of the alkynoic acid, the incorporation of [1-¹⁴C]acetate into hydrocarbon was inhibited 93%, the incorporation of [9,10-³H]oleate into hydrocarbon was inhibited 64%, and the incorporation of [1-¹⁴C]acetate into total internal lipid was inhibited 65%. Partially purified FAS was inhibited 50% and 95% at 15 μM and 40 μM, respectively, of the alkynoic acid. These results show that 2-octadecynoate inhibits hydrocarbon biosynthesis in the housefly by inhibiting FAS, and the in vivo data suggest that the elongation of 18:1 to longer chain fatty acids is also inhibited.     

Identification of the substrate recognition region in the Δ6-fatty acid and Δ8-sphingolipid desaturase by fusion mutagenesis

Δ⁸-sphingolipid desaturase and Δ⁶-fatty acid desaturase share high protein sequence identity. Thus, it has been hypothesized that Δ⁶-fatty acid desaturase is derived from Δ⁸-sphingolipid desaturase; however, there is no direct proof. The substrate recognition regions of Δ⁶-fatty acid desaturase and Δ⁸-sphingolipid desaturase, which aid in understanding the evolution of these two enzymes, have not been reported. A blackcurrant Δ⁶-fatty acid desaturase and a Δ⁸-sphingolipid desaturase gene, RnD6C and RnD8A, respectively, share more than 80 % identity in their coding protein sequences. In this study, a set of fusion genes of RnD6C and RnD8A were constructed and expressed in yeast. The Δ⁶- and Δ⁸-desaturase activities of the fusion proteins were characterized. Our results indicated that (1) the exchange of the C-terminal 172 amino acid residues can lead to a significant decrease in both desaturase activities; (2) amino acid residues 114–174, 206–257, and 258–276 played important roles in Δ⁶-substrate recognition, and the last two regions were crucial for Δ⁸-substrate recognition; and (3) amino acid residues 114–276 of Δ⁶-fatty acid desaturase contained the substrate recognition site(s) responsible for discrimination between ceramide (a substrate of Δ⁸-sphingolipid desaturase) and acyl-PC (a substrate of Δ⁶-fatty acid desaturase). Substituting the amino acid residues 114-276 of RnD8A with those of RnD6C resulted in a gain of Δ⁶-desaturase activity in the fusion protein but a loss in Δ⁸-sphingolipid desaturase activity. In conclusion, several regions important for the substrate recognition of Δ8-sphingolipid desaturase and Δ⁶-fatty acid desaturase were identified, which provide clues in understanding the relationship between the structure and function in desaturases.     

Adenylate cyclase orthologues in two filamentous entomopathogens contribute differentially to growth,conidiation, pathogenicity,and multistress responses

Adenylate cyclase (AC) is a core element of cAMP signalling network. Here we show functional diversity and differentiation of Beauveria bassiana AC (BbAC) and Metarhizium robertsii AC (MrAC). Severe growth defects occurred in ΔBbAC and ΔMrAC grown on nutrition-rich SDAY and several minimal media but were largely alleviated by adding cAMP to SDAY. Conidial yield increased greatly in ΔBbAC but decreased in ΔMrAC. During colony growth, ΔBbAC was highly sensitive to oxidation, high osmolarity, cell wall perturbation, carbendazim fungicide, Mn²⁺, Zn²⁺, Fe³⁺, and EDTA but more tolerant to Cu²⁺ while ΔMrAC showed higher osmotolerance, decreased sensitivity to Fe³⁺, and null response to carbendazim or cell wall stress despite similar responses to oxidation and other metal ions. Conidial UV-B resistance decreased by 32 % in ΔBbAC and 22 % in ΔMrAC despite little change in their theromotolerance. Median lethal time (LT₅₀) estimates of ΔBbAC and ΔMrAC against susceptible insects were 10.9 and 1.4 d longer than those from wild-type strains respectively. All the phenotypic changes were restored to wild-type levels by each gene complementation. Taken together, BbAC and MrAC regulated differentially conidiation, pathogenicity, and multistress responses in B. bassiana and M. robertsii, thereby making different contributions to their biocontrol potential.     

Two novel Physcomitrella patens fatty acid elongases (ELOs): identification and functional characterization

The lower plant Physcomitrella patens synthesizes several long-chain polyunsaturated fatty acids (LC-PUFAs) by a series of desaturation and elongation reactions. In the present study, the full-length cDNAs for two novel fatty acid elongases designated PpELO1 and PpELO2 were isolated from P. patens using a PCR-based cloning strategy. These cDNAs encoding proteins of 335 and 280 amino acids with predicted molecular masses of 38.7 and 32.9 kDa, respectively, are predicted to contain seven transmembrane domains with a possible localization in the subcellular endoplasmic reticulum. Sequence comparisons and phylogenetic analysis revealed that they are closely related to other LC-PUFA elongases of the lower eukaryotes such as the Δ⁵- and Δ⁶-elongases of Marchantia polymorpha as well as the Δ⁶-elongase of P. patens. Heterologous expression of the PpELO1 in Saccharomyces cerevisiae led to the elongation of Δ⁹-, Δ⁶-C₁₈, and Δ⁵-C₂₀ LC-PUFAs, whereas only Δ⁹- and Δ⁶-C₁₈ LC-PUFA substrates were used by PpELO2. Chimeric proteins were constructed to identify the amino acid regions most likely to be involved in the determination of the fatty acid substrate specificity. The expression of eight chimeric proteins in yeast revealed that substitution of the C-terminal 50 amino acids from PpELO1 into PpELO2 resulted in a high specificity for C₂₀ fatty acid substrates. As a result, we suggest that the C-terminal region of PpELO1 is sufficient for C₂₀ substrate elongation. Overall, these results provide important insights into the structural basis for substrate specificity of PUFA-generating ELO enzymes.     

(Na+,K+)-ATPase of mammalian brain: Effects of temperature on cation and ATP interactions regulating phosphatase activity

The effects of temperature on interactions between univalent cations or ATP and the p-nitrophenylphosphatase activity associated with brain (Na⁺,K⁺)-ATPase were examined. The apparent affinity for K⁺ activation under conditions favoring the moderate affinity site was temperature dependent, increasing with decreasing temperature. A comparison of univalent cations showed that the negative apparent ΔH and ΔS for cation binding increased with increasing apparent cation affinity. In contrast to the case with the moderate affinity sites, apparent affinity for the high affinity K⁺ site was independent of temperature. As temperature decreased, properties of moderate affinity site binding approached those of the high affinity site. The temperature dependence of ATP inhibition was opposite to that for K⁺ activation, with positive apparent ΔH and ΔS. The apparent ΔH and ΔS for cation binding approached those for the overall conformational change to K⁺-sensitive enzyme as cation affinity increased. These data suggest that E2, the K⁺-sensitive form of (Na⁺,K⁺)-ATPase, is stabilized by forces that require a decrease in entropy, explaining the predominant existence of E1 at physiologic temperatures. A conformational change leading to stabilization of E2 at higher temperatures can be produced by binding of univalent cations to a moderate affinity, presumably intracellular, site. This effect is counteracted by ATP. ATP also appears to alter the selectivity of this site to favor Na⁺ over K⁺ binding.     

Further Characteristics of the ATP-Stimulated Uptake of Calcium into Chromaffin Granules

Abstract: The ATP-stimulated uptake of ⁴⁵Ca²⁺ [and [³H](-)-noradrenaline ([³H]NA)] into chromaffin granules and that into mitochondria are driven by a protonic gradient ΔμH⁺, composed of the components ΔpH (concentration gradient of protons) and ΔΨ(electrical potential difference). The granular ATPase pumps protons into the matrix (ΔpH inside acid, ΔΨ positive), but the mitochondrial ATPase ejects protons from the matrix (ΔpH alkaline, ΔΨ negative inside). To show different driving forces of uptake, the rate of the ATP-stimulated uptake of ⁴⁵Ca²⁺ (and [³H]NA) into chromaffin granules was compared with the rate of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into mitochondria (adrenomedullary or rat liver). In the presence of nitrate, the rate of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into chromaffin granules is higher than in the presence of acetate, because the lyotropic anion nitrate stimulates the granular ATPase and increases ΔpH (acid inside). Compared with nitrate, the rate of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into mitochondria is higher in the presence of the proton-carrying anion acetate, which, after permeation, provides protons for ejection by the ATPase. In the absence of ATP, a valinomycin-mediated potassium influx (ΔΨ inside positive) stimulates the granular uptake of [³H]NA, which has an electrogenic component, but not the granular uptake of ⁴⁵Ca²⁺, which is electroneutral. The electrogenic uptake of ⁴⁵Ca²⁺ into mitochondria is stimulated by a valinomycin-mediated potassium efflux (ΔΨ negative inside). The ATP-stimulated uptake of ⁴⁵Ca²⁺ into chromaffin granules is sensitive to ruthenium red, suggesting a carrier-mediated mechanism of uptake, and it is sensitive to atractyloside, indicating the simultaneous uptake of ATP. After collapse of ΔpH by ammonia, the ATP-stimulated uptake of ⁴⁵Ca²⁺ into chromaffin granules is abolished, but not that into mitochondria. In the presence of ammonia, the rate of the ATP-stimulated uptake of [³H]NA is very low, and an ATP-independent uptake of ⁴⁵Ca²⁺ into chromaffin granules is observed which is similar to the ATP-independent Ca²⁺/Na⁺ exchange at the granular membrane.     

A novel type of energetics in a marine alkali-tolerant bacterium: Δμ-Na-driven motility and sodium cycle

Motility of a marine alkali-tolerant bacterium, Vibrio alginolyticus, can be observed in the presence of high concentrations of a protonophorous uncoupler, CCCP. Motility in the CCCP-containing media is completely inhibited by decrease in extracellular [Na⁺] or by monensin-induced increase in intracellular [Na⁺]. A mutant has been selected that grows only in media supplemented with a substrate such as acetate requiring no Δ$\text{μ}\text{-}$_Na to be transported into the cell. Motility of the mutant was found to be completely inhibited by CCCP. Cyanide, CCCP and vanadate added separately or in twos inhibit motility only partially. The three poisons added together completely paralyse the cells. In this inhibitor cocktail, arsenate can substitute for CCCP + vanadate; cyanide can be replaced by anaerobiosis. It is concluded that (i) Δ$\text{μ}\text{-}$_Na rather than Δ$\text{μ}\text{-}$_NH powers the flagellar motor of V. alginolyticus in the presence of CCCP, and (ii) in addition to the Na⁺-motive respiratory chain [Tokuda, H. and Unemoto, T. (1982) J. Biol. Chem. 257, 10007–10014] there is a vanadate and arsenate-sensitive oxygen-independent mechanism of Δ$\text{μ}$_Na generation, presumably an ion-motive ATPase. A suggestion is put forward that circulation of Na⁺ can replace that of H⁺ in V. alginolyticus, Δ$\text{μ}\text{-}$_Na being formed by the Na⁺-motive respiratory chain and utilized by Na⁺-solute symporters, the Na⁺-driven flagellar motor and maybe by a reverse ion-motive ATPase.