Proof for a nonproteinaceous calcium-selective channel in Escherichia coli by total synthesis from (R)-3-hydroxybutanoic acid and inorganic polyphosphate

Traditionally, the structure and properties of natural products have been determined by total synthesis and comparison with authentic samples. We have now applied this procedure to the first nonproteinaceous ion channel, isolated from bacterial plasma membranes, and consisting of a complex of poly(3-hydroxybutyrate) and calcium polyphosphate. To this end, we have now synthesized the 128-mer of hydroxybutanoic acid and prepared a complex with inorganic calcium polyphosphate (average 65-mer), which was incorporated into a planar lipid bilayer of synthetic phospholipids. We herewith present data that demonstrate unambiguously that the completely synthetic complex forms channels that are indistinguishable in their voltage-dependent conductance, in their selectivity for divalent cations, and in their blocking behavior (by La3+) from channels isolated from Escherichia coli. The implications of our finding for prebiotic chemistry, biochemistry, and biology are discussed.

Adenoviral gene transfer of Caenorhabditis elegans n−3 fatty acid desaturase optimizes fatty acid composition in mammalian cells

Omega−3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and have been shown to exert many preventive and therapeutic actions. The amount of n−3 PUFAs is insufficient in most Western people, whereas the level of n−6 PUFAs is relatively too high, with an n−6/n−3 ratio of >18. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations of n−3 PUFAs in mammals relies on chronic dietary intake of fat rich in n−3 PUFAs, because mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n−3 PUFAs or to convert n−6 to n−3 PUFAs. Here we report that adenovirus-mediated introduction of the Caenorhabditis elegans fat-1 gene encoding an n−3 fatty acid desaturase into mammalian cells can quickly and effectively elevate the cellular n−3 PUFA contents and dramatically balance the ratio of n−6/n−3 PUFAs. Heterologous expression of the fat-1 gene in rat cardiac myocytes rendered cells capable of converting various n−6 PUFAs to the corresponding n−3 PUFAs, and changed the n−6/n−3 ratio from about 15:1 to 1:1. In addition, an eicosanoid derived from n−6 PUFA (i.e., arachidonic acid) was reduced significantly in the transgenic cells. This study demonstrates an effective approach to modifying fatty acid composition of mammalian cells and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.

An in Vitro System from Maize Seedlings for Tryptophan-Independent Indole-3-Acetic Acid Biosynthesis1

The enzymatic synthesis of indole-3-acetic acid (IAA) from indole by an in vitro preparation from maize (Zea mays L.) that does not use tryptophan (Trp) as an intermediate is described. Light-grown seedlings of normal maize and the maize mutant orange pericarp were shown to contain the necessary enzymes to convert [14C]indole to IAA. The reaction was not inhibited by unlabeled Trp and neither [14C]Trp nor [14C]serine substituted for [14C]indole in this in vitro system. The reaction had a pH optimum greater than 8.0, required a reducing environment, and had an oxidation potential near that of ascorbate. The results obtained with this in vitro enzyme preparation provide strong, additional evidence for the presence of a Trp-independent IAA biosynthesis pathway in plants.

Metabolism of Indole-3-Acetic Acid in Arabidopsis1

The metabolism of indole-3-acetic acid (IAA) was investigated in 14-d-old Arabidopsis plants grown in liquid culture. After ruling out metabolites formed as an effect of nonsterile conditions, high-level feeding, and spontaneous interconversions, a simple metabolic pattern emerged. Oxindole-3-acetic acid (OxIAA), OxIAA conjugated to a hexose moiety via the carboxyl group, and the conjugates indole-3-acetyl aspartic acid (IAAsp) and indole-3-acetyl glutamate (IAGlu) were identified by mass spectrometry as primary products of IAA fed to the plants. Refeeding experiments demonstrated that none of these conjugates could be hydrolyzed back to IAA to any measurable extent at this developmental stage. IAAsp was further oxidized, especially when high levels of IAA were fed into the system, yielding OxIAAsp and OH-IAAsp. This contrasted with the metabolic fate of IAGlu, since that conjugate was not further metabolized. At IAA concentrations below 0.5 μm, most of the supplied IAA was metabolized via the OxIAA pathway, whereas only a minor portion was conjugated. However, increasing the IAA concentrations to 5 μm drastically altered the metabolic pattern, with marked induction of conjugation to IAAsp and IAGlu. This investigation used concentrations for feeding experiments that were near endogenous levels, showing that the metabolic pathways controlling the IAA pool size in Arabidopsis are limited and, therefore, make good targets for mutant screens provided that precautions are taken to avoid inducing artificial metabolism.

Indole-3-Acetic Acid Controls Cambial Growth in Scots Pine by Positional Signaling1

The vascular cambium produces secondary xylem and phloem in plants and is responsible for wood formation in forest trees. In this study we used a microscale mass-spectrometry technique coupled with cryosectioning to visualize the radial concentration gradient of endogenous indole-3-acetic acid (IAA) across the cambial meristem and the differentiating derivatives in Scots pine (Pinus sylvestris L.) trees that had different rates of cambial growth. This approach allowed us to investigate the relationship between growth rate and the concentration of endogenous IAA in the dividing cells. We also tested the hypothesis that IAA is a positional signal in xylem development (C. Uggla, T. Moritz, G. Sandberg, B. Sundberg [1996] Proc Natl Acad Sci USA 93: 9282–9286). This idea postulates that the width of the radial concentration gradient of IAA regulates the radial number of dividing cells in the cambial meristem, which is an important component for determining cambial growth rate. The relationship between IAA concentration in the dividing cells and growth rate was poor, although the highest IAA concentration was observed in the fastest-growing cambia. The radial width of the IAA concentration gradient showed a strong correlation with cambial growth rate. The results indicate that IAA gives positional information in plants.

Synthesis of medium-chain-length polyhydroxyalkanoates in Arabidopsis thaliana using intermediates of peroxisomal fatty acid β-oxidation

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the β-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the β-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.

Characterization of Transgenic Tobacco with an Increased α-Linolenic Acid Level1

Microsomal ω-3 fatty acid desaturase catalyzes the conversion of 18:2 (linoleic acid) to 18:3 (α-linolenic acid) in phospholipids, which are the main constituents of extrachloroplast membranes. Transgenic tobacco (Nicotiana tabacum) plants with increased 18:3 contents (designated SIIn plants) were produced through the introduction of a construct with the tobacco microsomal ω-3 fatty acid desaturase gene under the control of the highly efficient promoter containing the E12Ω sequence. 18:3 contents in the SIIn plants were increased by about 40% in roots and by about 10% in leaves compared with the control plants. With regard to growth at 15°C and 25°C and the ability to tolerate chilling at 1°C and 5°C, there were no discernible differences between the SIIn and the control plants. Freezing tolerance in leaves and roots, which was assessed by electrolyte leakage, was almost the same between the SIIn and the control plants. The fluidity of plasma membrane from the SIIn plants was almost the same as that of the control plants. These results indicate that an increase in the 18:3 level in phospholipids is not directly involved in compensation for the diminishment in growth or membrane properties observed under low temperatures.

Two auxin-responsive domains interact positively to induce expression of the early indoleacetic acid-inducible gene PS-IAA4/5.

The plant growth hormone indole-3-acetic acid (IAA) transcriptionally activates expression of several genes in plants. We have previously identified a 164-bp promoter region (-318 to -154) in the PS-IAA4/5 gene that confers IAA inducibility. Linker-scanning mutagenesis across the region has identified two positive domains: domain A (48 bp; -203 to -156) and domain B (44 bp; -299 to -256), responsible for transcriptional activation of PS-IAA4/5 by IAA. Domain A contains the highly conserved sequence 5'-TGTCCCAT-3' found among various IAA-inducible genes and behaves as the major auxin-responsive element. Domain B functions as an enhancer element which may also contain a less efficient auxin-responsive element. The two domains act cooperatively to stimulate transcription; however, tetramerization of domain A or B compensates for the loss of A or B function. The two domains can also mediate IAA-induced transcription from the heterologous cauliflower mosaic virus 35S promoter (-73 to +1). In vivo competition experiments with icosamers of domain A or B show that the domains interact specifically and with different affinities to low abundance, positive transcription factor(s). A model for transcriptional activation of PS-IAA4/5 by IAA is discussed.

Extracellular HIV-1 virus protein R causes a large inward current and cell death in cultured hippocampal neurons: Implications for AIDS pathology

The small HIV-1 accessory protein Vpr (virus protein R) is a multifunctional protein that is present in the serum and cerebrospinal fluid of AIDS patients. We previously showed that Vpr can form cation-selective ion channels across planar lipid bilayers, introducing the possibility that, if incorporated into the membranes of living cells, Vpr might form ion channels and consequently perturb the maintained ionic gradient. In this study, we demonstrate, by a variety of approaches, that Vpr added extracellularly to intact cells does indeed form ion channels. We use confocal laser scanning microscopy to examine the subcellular localization of fluorescently labeled Vpr. Plasmalemma depolarization and damage are examined using the anionic potential-sensitive dye bis(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI), respectively, and the effect of Vpr on whole-cell current is demonstrated directly by using the patch-clamp technique. We show that recombinant purified extracellular Vpr associates with the plasmalemma of hippocampal neurons to cause a large inward cation current and depolarization of the plasmalemma, eventually resulting in cell death. Thus, we demonstrate a physiological action of extracellular Vpr and present its mechanistic basis. These findings may have important implications for neuropathologies in AIDS patients who possess significant amounts of Vpr in the cerebrospinal fluid.

trans/cis (Z/E) photoisomerization of the chromophore of photoactive yellow protein is not a prerequisite for the initiation of the photocycle of this photoreceptor protein

The chromophore of photoactive yellow protein (PYP) (i.e., 4-hydroxycinnamic acid) has been replaced by an analogue with a triple bond, rather than a double bond (by using 4-hydroxyphenylpropiolic acid in the reconstitution, yielding hybrid I) and by a “locked” chromophore (through reconstitution with 7-hydroxycoumarin-3-carboxylic acid, in which a covalent bridge is present across the vinyl bond, resulting in hybrid II). These hybrids absorb maximally at 464 and 443 nm, respectively, which indicates that in both hybrids the deprotonated chromophore does fit into the chromophore-binding pocket. Because the triple bond cannot undergo cis/trans (or E/Z) photoisomerization and because of the presence of the lock across the vinyl double bond in hybrid II, it was predicted that these two hybrids would not be able to photocycle. Surprisingly, both are able. We have demonstrated this ability by making use of transient absorption, low-temperature absorption, and Fourier-transform infrared (FTIR) spectroscopy. Both hybrids, upon photoexcitation, display authentic photocycle signals in terms of a red-shifted intermediate; hybrid I, in addition, goes through a blue-shifted-like intermediate state, with very slow kinetics. We interpret these results as further evidence that rotation of the carbonyl group of the thioester-linked chromophore of PYP, proposed in a previous FTIR study and visualized in recent time-resolved x-ray diffraction experiments, is of critical importance for photoactivation of PYP.

High temperature promotes auxin-mediated hypocotyl elongation in Arabidopsis

Physiological studies with excised stem segments have implicated the plant hormone indole-3-acetic acid (IAA or auxin) in the regulation of cell elongation. Supporting evidence from intact plants has been somewhat more difficult to obtain, however. Here, we report the identification and characterization of an auxin-mediated cell elongation growth response in Arabidopsis thaliana. When grown in the light at high temperature (29°C), Arabidopsis seedlings exhibit dramatic hypocotyl elongation compared with seedlings grown at 20°C. This temperature-dependent growth response is sharply reduced by mutations in the auxin response or transport pathways and in seedlings containing reduced levels of free IAA. In contrast, mutants deficient in gibberellin and abscisic acid biosynthesis or in ethylene response are unaffected. Furthermore, we detect a corresponding increase in the level of free IAA in seedlings grown at high temperature, suggesting that temperature regulates auxin synthesis or catabolism to mediate this growth response. Consistent with this possibility, high temperature also stimulates other auxin-mediated processes including auxin-inducible gene expression. Based on these results, we propose that growth at high temperature promotes an increase in auxin levels resulting in increased hypocotyl elongation. These results strongly support the contention that endogenous auxin promotes cell elongation in intact plants.

Arrest of neuronal migration by excitatory amino acids in hamster developing brain

The influence of the excitotoxic cascade on the developing brain was investigated using ibotenate, a glutamatergic agonist of both N-methyl-d-aspartate (NMDA) ionotropic receptors and metabotropic receptors. Injected in the neopallium of the golden hamster at the time of production of neurons normally destined for layers IV, III, and II, ibotenate induces arrests of migrating neurons at different distances from the germinative zone within the radial migratory corridors. The resulting cytoarchitectonic patterns include periventricular nodular heterotopias, subcortical band heterotopias, and intracortical arrests of migrating neurons. The radial glial cells and the extracellular matrix are free of detectable damage that could suggest a defect in their guiding role. The migration disorders are prevented by coinjection of dl-2-amino-7-phosphoheptanoic acid, an NMDA ionotropic antagonist, but are not prevented by coinjection of l(+)-2-amino-3-phosphonopropionic acid, a metabotropic antagonist. This implies that an excess of ionic influx through the NMDA channels of neurons alters the metabolic pathways supporting neuronal migration. Ibotenate, a unique molecular trigger of the excitotoxic cascade, produces a wide spectrum of abnormal neuronal migration patterns recognized in mammals, including the neocortical deviations encountered in the human brain.

Identification in mice of two isoforms of the cysteinyl leukotriene 1 receptor that result from alternative splicing

Two classes of human G protein-coupled receptors, cysteinyl leukotriene 1 (CysLT1) and CysLT2 receptors, recently have been characterized and cloned. Because the CysLT1 receptor blockers are effective in treating human bronchial asthma and the mouse is often used to model human diseases, we isolated the mouse CysLT1 receptor from a mouse lung cDNA library and found two isoforms. A short isoform cDNA containing two exons encodes a polypeptide of 339 aa with 87.3% amino acid identity to the human CysLT1 receptor. A long isoform has two additional exons and an in-frame upstream start codon resulting in a 13-aa extension at the N terminus. Northern blot analysis revealed that the mouse CysLT1 receptor mRNA is expressed in lung and skin; and reverse transcription–PCR showed wide expression of the long isoform with the strongest presence in lung and skin. The gene for the mouse CysLT1 receptor was mapped to band XD. Leukotriene (LT) D4 induced intracellular calcium mobilization in Chinese hamster ovary cells stably expressing either isoform of the mouse CysLT1 receptor cDNA. This agonist effect of LTD4 was fully inhibited by the CysLT1 receptor antagonist, MK-571. Microsomal membranes from each transformant showed a single class of binding sites for [3H]LTD4; and the binding was blocked by unlabeled LTs, with the rank order of affinities being LTD4 >> LTE4 = LTC4 >> LTB4. Thus, the dominant mouse isoform with the N-terminal amino acid extension encoded by an additional exon has the same ligand response profile as the spliced form and the human receptor.

Brain 5α-dihydroprogesterone and allopregnanolone synthesis in a mouse model of protracted social isolation

Allopregnanolone (ALLO), is a brain endogenous neurosteroid that binds with high affinity to γ-aminobutyric acid type A (GABAA) receptors and positively modulates the action of GABA at these receptors. Unlike ALLO, 5α-dihydroprogesterone (5α-DHP) binds with high affinity to intracellular progesterone receptors that regulate DNA transcription. To investigate the physiological roles of ALLO and 5α-DHP synthesized in brain, we have adopted a mouse model involving protracted social isolation. In the frontal cortex of mice, socially isolated for 6 weeks, both neurosteroids were decreased by approximately 50%. After administration of (17β)-17-(bis-1-methyl amino carbonyl) androstane-3,5-diene-3-carboxylic acid (SKF105,111), an inhibitor of the enzyme (5α-reductase Type I and II) that converts progesterone into 5α-DHP, the ALLO and 5α-DHP content of frontal cortex of both group-housed and socially isolated mice decreased exponentially to 10%–20% of control values in about 30 min. The fractional rate constants (k h−1) of ALLO and 5α-DHP decline multiplied by the ALLO and 5α-DHP concentrations at any given steady-state estimate the rate of synthesis required to maintain that steady state. After 6 weeks of social isolation, ALLO and 5α-DHP biosynthesis rates were decreased to 30% of the values calculated in group-housed mice. Moreover, in socially isolated mice, the expression of 5α-reductase Type I mRNA and protein was approximately 50% lower than in group-housed mice whereas 3α-hydroxysteroid oxidoreductase mRNA expression was equal in the two groups. Protracted social isolation in mice may provide a model to investigate whether 5α-DHP by a genomic action, and ALLO by a nongenomic mechanism down-regulate the action of drugs acting as agonists, partial agonists, or positive allosteric modulators of the benzodiazepine recognition sites expressed by GABAA receptors.

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content, Assimilatory Charge, and Mesophyll Conductance in Leaves1

The content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Et; EC 4.1.1.39) measured in different-aged leaves of sunflower (Helianthus annuus) and other plants grown under different light intensities, varied from 2 to 75 μmol active sites m−2. Mesophyll conductance (μ) was measured under 1.5% O2, as well as postillumination CO2 uptake (assimilatory charge, a gas-exchange measure of the ribulose-1,5-bisphosphate pool). The dependence of μ on Et saturated at Et = 30 μmol active sites m−2 and μ = 11 mm s−1 in high-light-grown leaves. In low-light-grown leaves the dependence tended toward saturation at similar Et but reached a μ of only 6 to 8 mm s−1. μ was proportional to the assimilatory charge, with the proportionality constant (specific carboxylation efficiency) between 0.04 and 0.075 μm−1 s−1. Our data show that the saturation of the relationship between Et and μ is caused by three limiting components: (a) the physical diffusion resistance (a minor limitation), (b) less than full activation of Rubisco (related to Rubisco activase and the slower diffusibility of Rubisco at high protein concentrations in the stroma), and (c) chloroplast metabolites, especially 3-phosphoglyceric acid and free inorganic phosphate, which control the reaction kinetics of ribulose-1,5-bisphosphate carboxylation by competitive binding to active sites.