1-O-β-d-glucopyranosyleucommiol,an iridoid glucoside from Aucuba japonica

Eucommiol and a new eucommiol glucoside (eucommioside II) were isolated from Aucuba japonica, besides known aucubin. The structure of eucommioside     

Distribution of different forms of sterols in three cellular subfractions of Calendula officinalis leaves

From Calendula officinalis leaves three cellular subtractions (mitochondrial, Golgi membranes and microsomal) were obtained and enzymatically characterized. The contents of Δ⁰, Δ⁵, Δ⁷, Δ^{5, 22} sterols, as well as those of 24-methylenecholesterol and clerosterol, in the free and bound in the form of esters, glucosides and acylated glucosides were determined in these fractions. The results revealed the predominance of free sterols in the microsomal fraction, of esters in the mitochondrial fraction and of steryl glucosides and acylated glucosides in the Golgi fraction.     

Cloning and characterization of a Δ9-desaturase gene of the Antarctic fish Chionodraco hamatus and Trematomus bernacchii

Chionodraco hamatus and Trematomus bernacchii are perciforms, members of the fish suborder Notothenioidei that live in the Antarctic Ocean and experience very cold and persistent environmental temperature. These fish have biochemical and molecular features that allow them to live at these extreme cold temperatures. Fine tuning of the level of unsaturated fatty acids content in membrane is a key mechanism of living organisms to adapt to cold and high temperatures. Desaturases are key enzymes that synthesize unsaturated fatty acyl-CoAs from saturated fatty acids. We cloned and sequenced a Δ⁹-desaturase gene and its cDNA of C. hamatus, and the cDNA of T. bernacchii. The coded proteins are virtually identical and share homology to other Δ⁹-desaturase fish sequences. These proteins contain, in the first trans-membrane domain, two cysteine residues that may form a disulfur bond present in the corresponding membrane region of Δ⁹-desaturase proteins of other Antarctic fish but not in Eleginops maclovinus that experiences higher environmental temperatures and in all other Δ⁹-desaturase genes of mammals present in data bases. C. hamatus Δ⁹-desaturase gene complements a Saccharomyces cerevisiae mutant lacking Δ⁹-desaturase (Ole1) gene. Analysis of sequence homology of the trans-membrane domains of Δ⁹-desaturase and the cytoplasmic region of the same proteins of Antarctic fish, non-Antarctic fish and mammals suggest that the significant differences found in the homologous sequences of the first trans-membrane domain may be due to the specific lipid content of their membrane.     

(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.     

Biosynthesis of the iridoids aucubin antirrinoside from 8-epi-deoxyloganic acid

²H NMR spectroscopy shows that 8-epi-deoxy[6,7,8,10-²H₄]loganic acid is efficiently incorporated into aucubin in Plantago major and Scrophularia racemosa, and into antirrinoside in Antirrhinum majus. Deoxyloganic acid produced no observable incorporation in these species.     

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.     

Microorganisms and nematodes increase levels of secondary metabolites in roots and root exudates of Plantago lanceolata

Plant secondary metabolites play an important role in constitutive and inducible direct defense of plants against their natural enemies. While induction of defense by aboveground pathogens and herbivores is well-studied, induction by belowground organisms is less explored. Here, we examine whether soil microorganisms and nematodes can induce changes in levels of the secondary metabolites aucubin and catalpol (iridoid glycosides, IG) in roots and root exudates of two full-sib families of Plantago lanceolata originating from lines selected for low and high constitutive levels of IG in leaves. Addition of soil microorganisms enhanced the shoot and root biomass, and the concentration of aucubin in roots of both Plantago lines without affecting IG levels in the rhizosphere. By contrast, nematode addition tended to reduce the root biomass and enhanced the stalk biomass, and increased the levels of aucubin and catalpol in root exudates of both Plantago lines, without affecting root IG concentrations. The Plantago lines did not differ in constitutive levels of aucubin and total IG in roots, while the concentration of catalpol was slightly higher in roots of plants originally selected for low constitutive levels of IG in leaves. Root exudates of “high IG line” plants contained significantly higher levels of aucubin, which might be explained by their higher root biomass. We conclude that soil microorganisms can induce an increase of aucubin concentrations in the roots, whereas nematodes (probably plant feeders) lead to an enhancement of aucubin and catalpol levels in root exudates of P. lanceolata. A potential involvement of secondary metabolites in belowground interactions between plants and soil organisms is discussed.     

Ratiometric fluorescence measurements of membrane potential generated by yeast plasma membrane H-ATPase reconstituted into vesicles

Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Δψ) generated by the Schizosaccharomyces pombe plasma membrane H⁺-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Δψ because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Δψ at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H⁺-ATPase generates Δψ of about 160 mV on the vesicle membrane. The generated Δψ was stable at least over tens of minutes. An influence of the H⁺ membrane permeability on the Δψ buildup was demonstrated by manipulating the H⁺ permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H⁺-ATPase-mediated Δψ buildup.     

Enhanced production of poly(lactate-co-3-hydroxybutyrate) from xylose in engineered Escherichia coli overexpressing a galactitol transporter

Poly(lactate-co-3-hydroxybutyrate) (P(LA-co-3HB)) was previously produced from xylose in engineered Escherichia coli. The aim of this study was to increase the polymer productivity and LA fraction in P(LA-co-3HB) using two metabolic engineering approaches: (1) deletions of competing pathways to lactate production and (2) overexpression of a galactitol transporter (GatC), which contributes to the ATP-independent xylose uptake. Engineered E. coli mutants (ΔpflA, Δpta, ΔackA, ΔpoxB, Δdld, and a dual mutant; ΔpflA?+?Δdld) and their parent strain, BW25113, were grown on 20 g l^?1 xylose for P(LA-co-3HB) production. The single deletions of ΔpflA, Δpta, and Δdld increased the LA fraction (58–66 mol%) compared to BW25113 (56 mol%). In particular, the ΔpflA?+?Δdld strain produced P(LA-co-3HB) containing 73 mol% LA. Furthermore, GatC overexpression increased both polymer yields and LA fractions in ΔpflA, Δpta, and Δdld mutants, and BW25113. The ΔpflA?+?gatC strain achieved a productivity of 8.3 g l^?1, which was 72 % of the theoretical maximum yield. Thus, to eliminate limitation of the carbon source, higher concentration of xylose was fed. As a result, BW25113 harboring gatC grown on 40 g l^?1 xylose reached the highest P(LA-co-3HB) productivity of 14.4 g l^?1. On the other hand, the ΔpflA?+?Δdld strain grown on 30 g l^?1 xylose synthesized 6.4 g l^?1 P(LA-co-3HB) while maintaining the highest LA fraction (73 mol%). The results indicated the usefulness of GatC for enhanced production of P(LA-co-3HB) from xylose, and the gene deletions to upregulate the LA fraction in P(LA-co-3HB). The polymers obtained had weight-averaged molecular weights in the range of 34,000–114,000.     

Structure elucidation of eucommioside (2″-O-β-d-glucopyranosyl eucommiol) from Eucommia ulmoides

The isolation of eucommioside from Eucommia ulmoides is described. Chemical modifications and spectral evidence identify eucommioside as the 2″-O-β-d-glucopyranosyl derivative of eucommiol, a known cyclopentenoidtetrol previously isolated from the same plant.     

IGERS: Inferring Gibbs Energy Changes of Biochemical Reactions from Reaction Similarities

Mathematical analysis and modeling of biochemical reaction networks requires knowledge of the permitted directionality of reactions and membrane transport processes. This information can be gathered from the standard Gibbs energy changes (ΔG⁰) of reactions and the concentration ranges of their reactants. Currently, experimental ΔG⁰ values are not available for the vast majority of cellular biochemical processes. We propose what we believe to be a novel computational method to infer the unknown ΔG⁰ value of a reaction from the known ΔG⁰ value of the chemically most similar reaction. The chemical similarity of two arbitrary reactions is measured by the relative number (T) of co-occurring changes in the chemical attributes of their reactants. Testing our method across a validated reference set of 173 biochemical reactions with experimentally determined ΔG⁰ values, we found that a minimum reaction similarity of T = 0.6 is required to infer ΔG⁰ values with an error of <10 kJ/mol. Applying this criterion, our method allows us to assign ΔG⁰ values to 458 additional reactions of the BioPath database. We believe our approach permits us to minimize the number of ΔG⁰ measurements required for a full coverage of a given reaction network with reliable ΔG⁰ values.     

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.     

Inhibitory effects of alkyl gallate and its derivatives on fatty acid desaturation

Alkyl gallate, which is known as an antioxidant, intensively inhibited Δ5 and Δ6 desaturation in both rat liver microsomes and an arachidonic acid-producing fungus Mortierella alpina 1S-4. The rat liver microsomal Δ5 and Δ6 desaturases were inhibited by gallic acid esterified with alcohols with various numbers of carbons, suggesting that the necessary structure in an esterified alcohol for the inhibition is not so strict. Among the three hydroxy groups in gallic acid, the m-hydroxy group was shown to be the necessary structure. Kinetic analyses revealed that propyl gallate is a noncompetitive inhibitor of Δ5 desaturase (K_i = 2.6 · 10⁻⁵ M) and Δ6 desaturase (K_i = 1.7 · 10⁻⁴ M). These data indicate that alkyl gallate is a new type of desaturase inhibitor and different from known natural inhibitors, i.e., sesamin and curcumin.     

RecBCD- RecFOR-independent pathway of homologous recombination in Escherichia coli

The RecA protein is a key bacterial recombination enzyme that catalyzes pairing and strand exchange between homologous DNA duplexes. In Escherichia coli, RecA protein assembly on DNA is mediated either by the RecBCD or RecFOR protein complexes. Correspondingly, two recombination pathways, RecBCD and RecF (or RecFOR), are distinguished in E. coli. Inactivation of both pathways in recB(CD) recF(OR) mutants results in severe recombination deficiency. Here we describe a novel, RecBCD- RecFOR-independent (RecBFI) recombination pathway that is active in ΔrecBCD sbcB15 sbcC(D) ΔrecF(OR) mutants of E. coli. In transductional crosses, these mutants show only four-fold decrease of recombination frequency relative to the wild-type strain. At the same time they recombine 40- to 90-fold better than their sbcB⁺ sbcC⁺ and ΔsbcB sbcC counterparts. The RecBFI pathway strongly depends on recA, recJ and recQ gene functions, and moderately depends on recG and lexA functions. Inactivation of dinI, helD, recX, recN, radA, ruvABC and uvrD genes has a slight effect on RecBFI recombination. After exposure to UV and gamma irradiation, the ΔrecBCD sbcB15 sbcC ΔrecF mutants show moderately increased DNA repair proficiency relative to their sbcB⁺ sbcC⁺ and ΔsbcB sbcC counterparts. However, introduction of recA730 allele (encoding RecA protein with enhanced DNA binding properties) completely restores repair proficiency to ΔrecBCD sbcB15 sbcC ΔrecF mutants, but not to their sbcB⁺ sbcC⁺ and ΔsbcB sbcC derivatives. Fluorescence microscopy with UV-irradiated recA-gfp fusion mutants suggests that the kinetics of RecA filament formation might be slowed down in the RecBFI pathway. Inactivation of 3′-5′ exonucleases ExoVII, ExoIX and ExoX cannot activate the RecBFI pathway in ΔrecBCD ΔsbcB sbcC ΔrecF mutants. Taken together, our results show that the product of the sbcB15 allele is crucial for RecBFI pathway. Besides protecting 3′ overhangs, SbcB15 protein might play an additional, more active role in formation of the RecA filament.     

Sterols and fatty acids of the marine diatom biddulphia sinensis

Nine sterols, most showing Δ⁵- or Δ^5,22-unsaturation, were identified in the marine diatom Biddulphia sinensis. One sterol, cholesta-5,22E-dien-3β-ol, comprised 70–80% of the total sterols which is the first such predominance noted in a diatom. The only Δ⁷-sterol detected was cholest-7-en-3β-ol and this was a very minor component. A sterol showing unusual side-chain alkylation,23,24-dimethylcholesta-5,22E-dien-3β-ol, was identified for the first time in a diatom. Total fatty acids exhibited a predominance of Δ⁹- 16:1, 14:0, 20:5 and 16:0, typical of diatoms, although the proportions of these acids were found to vary with culture maturity. n-Heneicosahexaene was the major hydrocarbon together with a small amount of squalene.     

Marcus treatment of endergonic reactions: A commentary

Two forms of the equation for expression of the rate constant for electron transfer through a Marcus-type treatment are discussed. In the first (exergonic) form, the Arrhenius exponential term was replaced by its classical Marcus term; in the second (endergonic) form, the forward rate constant was replaced by the reverse rate constant (the forward rate constant in the exergonic direction), which was expanded to an equivalent Marcus term and multiplied by the equilibrium constant. When the classical Marcus treatment was used, these two forms of the rate equation give identical curves relating the logarithm of the rate constant to the driving force. The Marcus term for the relation between activation free-energy, ΔG^#, reorganization energy, λ, and driving force, ΔG^o, derived from parabolas for the reactant and product states, was identical when starting from exergonic or endergonic parabolas. Moser and colleagues introduced a quantum mechanical correction factor to the Marcus term in order to fit experimental data. When the same correction factor was applied in the treatment for the endergonic direction by Page and colleagues, a different curve was obtained from that found with the exergonic form. We show that the difference resulted from an algebraic error in development of the endergonic equation.     

Sapogenins of Yucca glauca seed pods

From the fruiting pods of Yucca glauca Nutt. devoid of seeds the following sapogenins have been isolated and identified: neo-tigogenin, hecogenin, gitogenin, manogenin, Δ⁹-manogenin and sarsasapogenin. A small amount of a material believed to be Δ²-desoxysarsasapogenin was also isolated and is apparently an artifact arising from the hydrolysis and extraction procedure. Manogenin and Δ⁹-manogenin have not been previously detected in Y. glauca, and Δ⁹-manogenin has not been reported in any Yucca species.     

Comparative study of the inhibition of sterol biosynthesis in Rubus fruticosus suspension cultures and zea mays seedlings by N-(1,5,9-trimethyldecyl)-4α,10-dimethyl-8-aza-trans-decal-3β-ol and derivatives

The nitrogen substituents present in tridemorph and fenpropimorph, which are systemic fungicides, have been linked to an 8-aza-bicyclic skeleton leading to N-(1,5,9-trimethyldecyl)-4α,10-dimethyl-8-aza-trans-decal-3β-ol and N-(3-(4-tert-butylphenyl-)2-methyl)-propyl-8-aza-4α,10-dimethyl-trans-decal-3β-ol respectively. The latter two compounds present in a stable molecule key structural elements of unstable C-8 and C-9 carbocationic high-energy intermediates which occur during the reactions catalysed by the Δ⁸ → Δ⁷-sterol isomerase and the cycloeucalenol-obtusifoliol isomerase, respectively. When given to either bramble cell suspension cultures or maize seedlings, they led to a spectacular accumulation of 9β,19-cyclopropyl sterols and were in that respect much more efficient than any known molecules and in particular than the N-benzyl decalin previously described which led to accumulation of Δ⁸-sterols. Surprisingly, treatment of the plant cells by the N-oxide derivatives of the N-benzyl decalin resulted in dramatic accumulation of Δ^8,14-sterols.     

The kinetics and mechanism of the reaction of 2′-deoxy-5′-guanosinemonophosphoric acid and the diaqua form of cis-platin

2′-Deoxy-5′-guanosinemonosphoric acid (B) reacts with cis-[Pt(NH₃)₂(OH₂)₂]²⁺ in two steps to form the cis-[Pt(NH₃)₂B₂]^y+ ion. In the first step 2′-d-5′- GMPH₂ reacts some ten times faster than 5′-GMPH₂ does. Rate constants, ΔH^#, ΔS^# and ΔV^# are very similar for the two bases in the second reaction. It is proposed that the product in the first step contains no water and is cis-[Pt(NH₃)₂B]^x+ in which the nucleobase is bidentate bonding through both N(7) of guanine and an oxygen atom of the phosphate group.