Trends in lipoplex physical properties dependent on cationic lipid structure, vehicle and complexation procedure do not correlate with biological activity

Using a group of structurally related cytofectins, the effects of different vehicle constituents and mixing techniques on the physical properties and biological activity of lipoplexes were systematically examined. Physical properties were examined using a combination of dye accessibility assays, centrifugation, gel electrophoresis and dynamic light scattering. Biological activity was examined using in vitro transfection. Lipoplexes were formulated using two injection vehicles commonly used for in vivo delivery (PBS pH 7.2 and 0.9% saline), and a sodium phosphate vehicle previously shown to enhance the biological activity of naked pDNA and lipoplex formulations. Phosphate was found to be unique in its effect on lipoplexes. Specifically, the accessible pDNA in lipoplexes formulated with cytofectins containing a γ-amine substitution in the headgroup was dependent on alkyl side chain length and sodium phosphate concentration, but the same effects were not observed when using cytofectins containing a β-OH headgroup substitution. The physicochemical features of the phosphate anion, which give rise to this effect in γ-amine cytofectins, were deduced using a series of phosphate analogs. The effects of the formulation vehicle on transfection were found to be cell type-dependent; however, of the formulation variables examined, the liposome/pDNA mixing method had the greatest effect on transgene expression in vitro. Thus, though predictive physical structure relationships involving the vehicle and cytofectin components of the lipoplex were uncovered, they did not extrapolate to trends in biological activity.

Mannose Inhibits Arabidopsis Germination via a Hexokinase-Mediated Step1

Low concentrations of the glucose (Glc) analog mannose (Man) inhibit germination of Arabidopsis seeds. Man is phosphorylated by hexokinase (HXK), but the absence of germination was not due to ATP or phosphate depletion. The addition of metabolizable sugars reversed the Man-mediated inhibition of germination. Carbohydrate-mediated regulation of gene expression involving a HXK-mediated pathway is known to be activated by Glc, Man, and other monosaccharides. Therefore, we investigated whether Man blocks germination through this system. By testing other Glc analogs, we found that 2-deoxyglucose, which, like Man, is phosphorylated by HXK, also blocked germination; no inhibition was observed with 6-deoxyglucose or 3-O-methylglucose, which are not substrates for HXK. Since these latter two sugars are taken up at a rate similar to that of Man, uptake is unlikely to be involved in the inhibition of germination. Furthermore, we show that mannoheptulose, a specific HXK inhibitor, restores germination of seeds grown in the presence of Man. We conclude that HXK is involved in the Man-mediated repression of germination of Arabidopsis seeds, possibly via energy depletion.

Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands

The crystal structures of the ligand-binding domain (LBD) of the vitamin D receptor complexed to 1α,25(OH)2D3 and the 20-epi analogs, MC1288 and KH1060, show that the protein conformation is identical, conferring a general character to the observation first made for retinoic acid receptor (RAR) that, for a given LBD, the agonist conformation is unique, the ligands adapting to the binding pocket. In all complexes, the A- to D-ring moieties of the ligands adopt the same conformation and form identical contacts with the protein. Differences are observed only for the 17β-aliphatic chains that adapt their conformation to anchor the 25-hydroxyl group to His-305 and His-397. The inverted geometry of the C20 methyl group induces different paths of the aliphatic chains. The ligands exhibit a low-energy conformation for MC1288 and a more strained conformation for the two others. KH1060 compensates this energy cost by additional contacts. Based on the present data, the explanation of the superagonist effect is to be found in higher stability and longer half-life of the active complex, thereby excluding different conformations of the ligand binding domain.

An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense

Plants contain RNA-dependent RNA polymerase (RdRP) activities that synthesize short cRNAs by using cellular or viral RNAs as templates. During studies of salicylic acid (SA)-induced resistance to viral pathogens, we recently found that the activity of a tobacco RdRP was increased in virus-infected or SA-treated plants. Biologically active SA analogs capable of activating plant defense response also induced the RdRP activity, whereas biologically inactive analogs did not. A tobacco RdRP gene, NtRDRP1, was isolated and found to be induced both by virus infection and by treatment with SA or its biologically active analogs. Tobacco lines deficient in the inducible RDRP activity were obtained by expressing antisense RNA for the NtRDRP1 gene in transgenic plants. When infected by tobacco mosaic virus, these transgenic plants accumulated significantly higher levels of viral RNA and developed more severe disease symptoms than wild-type plants. After infection by a strain of potato virus X that does not spread in wild-type tobacco plants, the transgenic NtRDRP1 antisense plants accumulated virus and developed symptoms not only locally in inoculated leaves but also systemically in upper uninoculated leaves. These results strongly suggest that inducible RdRP activity plays an important role in plant antiviral defense.

Three independent lines of evidence suggest retinoids as causal to schizophrenia

Retinoid dysregulation may be an important factor in the etiology of schizophrenia. This hypothesis is supported by three independent lines of evidence that triangulate on retinoid involvement in schizophrenia: (i) congenital anomalies similar to those caused by retinoid dysfunction are found in schizophrenics and their relatives; (ii) those loci that have been suggestively linked to schizophrenia are also the loci of the genes of the retinoid cascade (convergent loci); and (iii) the transcriptional activation of the dopamine D2 receptor and numerous schizophrenia candidate genes is regulated by retinoic acid. These findings suggest a close causal relationship between retinoids and the underlying pathophysiological defects in schizophrenia. This leads to specific strategies for linkage analyses in schizophrenia. In view of the heterodimeric nature of the retinoid nuclear receptor transcription factors, e.g., retinoid X receptor β at chromosome 6p21.3 and retinoic acid receptor β at 3p24.3, two-locus linkage models incorporating genes of the retinoid cascade and their heterodimeric partners, e.g., peroxisome proliferator-activated receptor α at chromosome 22q12-q13 or nuclear-related receptor 1 at chromosome 2q22-q23, are proposed. New treatment modalities using retinoid analogs to alter the downstream expression of the dopamine receptors and other genes that are targets of retinoid regulation, and that are thought to be involved in schizophrenia, are suggested.

Function and Substrate Specificity of the Gibberellin 3β-Hydroxylase Encoded by the Arabidopsis GA4 Gene1

cDNA corresponding to the GA4 gene of Arabidopsis thaliana L. (Heynh.) was expressed in Escherichia coli, from which cell lysates converted [14C]gibberellin (GA)9 and [14C]GA20 to radiolabeled GA4 and GA1, respectively, thereby confirming that GA4 encodes a GA 3β-hydroxylase. GA9 was the preferred substrate, with a Michaelis value of 1 μm compared with 15 μm for GA20. Hydroxylation of these GAs was regiospecific, with no indication of 2β-hydroxylation or 2,3-desaturation. The capacity of the recombinant enzyme to hydroxylate a range of other GA substrates was investigated. In general, the preferred substrates contained a polar bridge between C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated analogs. Therefore, no activity was detected using GA12-aldehyde, GA12, GA19, GA25, GA53, or GA44 as the open lactone (20-hydroxy-GA53), whereas GA15, GA24, and GA44 were hydroxylated to GA37, GA36, and GA38, respectively. The open lactone of GA15 (20-hydroxy-GA12) was hydroxylated but less efficiently than GA15. In contrast to the free acid, GA25 19,20-anhydride was 3β-hydroxylated to give GA13. 2,3-Didehydro-GA9 and GA5 were converted by recombinant GA4 to the corresponding epoxides 2,3-oxido-GA9 and GA6.

Effects of Sulfanilamide and Methotrexate on 13C Fluxes through the Glycine Decarboxylase/Serine Hydroxymethyltransferase Enzyme System in Arabidopsis1

In C3 plants large amounts of photorespiratory glycine (Gly) are converted to serine by the tetrahydrofolate (THF)-dependent activities of the Gly decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT). Using 13C nuclear magnetic resonance, we monitored the flux of carbon through the GDC/SHMT enzyme system in Arabidopsis thaliana (L.) Heynh. Columbia exposed to inhibitors of THF-synthesizing enzymes. Plants exposed for 96 h to sulfanilamide, a dihydropteroate synthase inhibitor, showed little reduction in flux through GDC/SHMT. Two other sulfonamide analogs were tested with similar results, although all three analogs competitively inhibited the partially purified enzyme. However, methotrexate or aminopterin, which are confirmed inhibitors of Arabidopsis dihydrofolate reductase, decreased the flux through the GDC/SHMT system by 60% after 48 h and by 100% in 96 h. The uptake of [α-13C]Gly was not inhibited by either drug class. The specificity of methotrexate action was shown by the ability of 5-formyl-THF to restore flux through the GDC/SHMT pathway in methotrexate-inhibited plants. The experiments with sulfonamides strongly suggest that the mitochondrial THF pool has a long half-life. The studies with methotrexate support the additional, critical role of dihydrofolate reductase in recycling THF oxidized in thymidylate synthesis.

Nucleotide Triphosphates Are Required for the Transport of Glycolate Oxidase into Peroxisomes1

All peroxisomal proteins are nuclear encoded, synthesized on free cytosolic ribosomes, and posttranslationally targeted to the organelle. We have used an in vitro assay to reconstitute protein import into pumpkin (Cucurbita pepo) glyoxysomes, a class of peroxisome found in the cotyledons of oilseed plants, to study the mechanisms involved in protein transport across peroxisome membranes. Results indicate that ATP hydrolysis is required for protein import into peroxisomes; nonhydrolyzable analogs of ATP could not substitute for this requirement. Nucleotide competition studies suggest that there may be a nucleotide binding site on a component of the translocation machinery. Peroxisomal protein import also was supported by GTP hydrolysis. Nonhydrolyzable analogs of GTP did not substitute in this process. Experiments to determine the cation specificity of the nucleotide requirement show that the Mg2+ salt was preferred over other divalent and monovalent cations. The role of a putative protonmotive force across the peroxisomal membrane was also examined. Although low concentrations of ionophores had no effect on protein import, relatively high concentrations of all ionophores tested consistently reduced the level of protein import by approximately 50%. This result suggests that a protonmotive force is not absolutely required for peroxisomal protein import.

Polypeptide signaling for plant defensive genes exhibits analogies to defense signaling in animals.

The activation of plant defensive genes in leaves of tomato plants in response to herbivore damage or mechanical wounding is mediated by a mobile 18-amino acid polypeptide signal called systemin. Systemin is derived from a larger, 200-amino acid precursor called prosystemin, similar to polypeptide hormones and soluble growth factors in animals. Systemin activates a lipid-based signaling cascade, also analogous to signaling systems found in animals. In plants, linolenic acid is released from membranes and is converted to the oxylipins phytodienoic acid and jasmonic acid through the octadecanoid pathway. Plant oxylipins are structural analogs of animal prostaglandins which are derived from arachidonic acid in response to various signals, including polypeptide factors. Constitutive overexpression of the prosystemin gene in transgenic tomato plants resulted in the overproduction of prosystemin and the abnormal release of systemin, conferring a constitutive overproduction of several systemic wound-response proteins (SWRPs). The data indicate that systemin is a master signal for defense against attacking herbivores. The same defensive proteins induced by wounding are synthesized in response to oligosaccharide elicitors that are generated in leaf cells in response to pathogen attacks. Inhibitors of the octadecanoid pathway, and a mutation that interrupts this pathway, block the induction of SWRPs by wounding, systemin, and oligosaccharide elicitors, indicating that the octadecanoid pathway is essential for the activation of defense genes by all of these signals. The tomato mutant line that is functionally deficient in the octadecanoid pathway is highly susceptible to attacks by Manduca sexta larvae. The similarities between the defense signaling pathway in tomato leaves and those of the defense signaling pathways of macrophages and mast cells of animals suggests that both the plant and animal pathways may have evolved from a common ancestral origin.

Direct evidence for modified solvent structure within the hydration shell of a hydrophobic amino acid.

Neutron scattering experiments are used to determine scattering profiles for aqueous solutions of hydrophobic and hydrophilic amino acid analogs. Solutions of hydrophobic solutes show a shift in the main diffraction peak to smaller angle as compared with pure water, whereas solutions of hydrophilic solutes do not. The same difference for solutions of hydrophobic and hydrophilic side chains is also predicted by molecular dynamics simulations. The neutron scattering curves of aqueous solutions of hydrophobic amino acids at room temperature are qualitatively similar to differences between the liquid molecular structure functions measured for ambient and supercooled water. The nonpolar solute-induced expansion of water structure reported here is also complementary to recent neutron experiments where compression of aqueous solvent structure has been observed at high salt concentration.

Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex.

Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide-MHC complexes have been less well characterized. We have used a complete set of singly substituted analogs of a mouse MHC class I, Kk-restricted peptide, influenza hemagglutinin (Ha)255-262, to address the binding specificity of this MHC molecule. Using the same peptide-MHC complexes we determined the fine specificity of two Ha255-262-specific, Kk-restricted T cells, and of a unique antibody, pSAN, specific for the same peptide-MHC complex. Independently, a model of the Ha255-262-Kk complex was generated through homology modeling and molecular mechanics refinement. The functional data and the model corroborated each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody.

Mice lacking the gene encoding tissue-type plasminogen activator show a selective interference with late-phase long-term potentiation in both Schaffer collateral and mossy fiber pathways.

The gene encoding tissue-type plasminogen activator (t-PA) is an immediate response gene, downstream from CREB-1 and other constitutively expressed transcription factors, which is induced in the hippocampus during the late phase of long-term potentiation (L-LTP). Mice in which the t-PA gene has been ablated (t-PA-/-) showed no gross anatomical, electrophysiological, sensory, or motor abnormalities but manifest a selective reduction in L-LTP in hippocampal slices in both the Schaffer collateral-CA1 and mossy fiber-CA3 pathways. t-PA-/- mice also exhibit reduced potentiation by cAMP analogs and D1/D5 agonists. By contrast, hippocampal-dependent learning and memory were not affected in these mice, whereas performance was impaired on two-way active avoidance, a striatum-dependent task. These results provide genetic evidence that t-PA is a downstream effector gene important for L-LTP and show that modest impairment of L-LTP in CA1 and CA3 does not result in hippocampus-dependent behavioral phenotypes.

1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease believed to be a model for the human disease multiple sclerosis (MS). Induced by immunizing B10.PL mice with myelin basic protein (MBP), EAE was completely prevented by the administration of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. 1,25-(OH)2D3 could also prevent the progression of EAE when administered at the appearance of the first disability symptoms. Withdrawal of 1,25-(OH)2D3 resulted in a resumption of the progression of EAE. Thus, the block by 1,25-(OH)2D3 is reversible. A deficiency of vitamin D resulted in an increased susceptibility to EAE. Thus, 1,25-(OH)2D3 or its analogs are potentially important for treatment of MS.

A mechanism for inducing plant development: the genesis of a specific inhibitor.

Parasitic strategies are widely distributed in the plant kingdom and frequently involve coupling parasite organogenesis with cues from the host. In Striga asiatica, for example, the cues that initiate the development of the host attachment organ, the haustorium, originate in the host and trigger the transition from vegetative to parasitic mode in the root meristem. This system therefore offers a unique opportunity to study the signals and mechanisms that control plant cell morphogenesis. Here we establish that the biological activity of structural analogs of the natural inducer displays a marked dependence on redox potential and suggest the existence of a semiquinone intermediate. Building on chemistry that exploits the energetics of such an intermediate, cyclopropyl-p-benzoquinone (CPBQ) is shown to be a specific inhibitor of haustorial development. These data are consistent with a model where haustorial development is initiated by the completion of a redox circuit.

Template-nucleated alanine-lysine helices are stabilized by position-dependent interactions between the lysine side chain and the helix barrel.

The helicity in water has been determined for several series of alanine-rich peptides that contain single lysine residues and that are N-terminally linked to a helix-inducing and reporting template termed Ac-Hel1. The helix-propagating constant for alanine (sAla value) that best fits the properties of these peptides lies in the range of 1.01-1.02, close to the value reported by Scheraga and coworkers [Wojcik, J., Altmann, K.-H. & Scheraga, H.A. (1990) Biopolymers 30, 121-134], but significantly lower than the value assigned by Baldwin and coworkers [Chakrabartty, A., Kortemme, T. & Baldwin, R.L. (1994) Protein Sci. 3,843-852]. From a study of conjugates Ac-Hel1-Ala(n)-Lys-Ala(m)-NH2 and analogs in which the methylene portion of the lysine side chain is truncated, we find that the unusual helical stability of Ala(n)Lys peptides is controlled primarily by interactions of the lysine side chain with the helix barrel, and only passively by the alanine matrix. Using 1H NMR spectroscopy, we observe nuclear Overhauser effect crosspeaks consistent with proton-proton contacts expected for these interactions.