Structural intermediates in the fusion‐associated transition of vesiculovirus glycoprotein

Vesiculoviruses enter cells by membrane fusion, driven by a large, low‐pH‐induced, conformational change in the fusion glycoprotein G that involves transition from a trimeric pre‐fusion toward a trimeric post‐fusion state via monomeric intermediates. Here, we present the structure of the G fusion protein at intermediate pH for two vesiculoviruses, vesicular stomatitis virus (VSV) and Chandipura virus (CHAV), which is responsible for deadly encephalopathies. First, a CHAV G crystal structure shows two intermediate conformations forming a flat dimer of heterodimers. On virions, electron microscopy (EM) and tomography reveal monomeric spikes similar to one of the crystal conformations. In solution, mass spectrometry shows dimers of G. Finally, mutations at a dimer interface, involving fusion domains associated in an antiparallel manner to form an intermolecular β‐sheet, affect G fusion properties. The location of the compensatory mutations restoring fusion activity strongly suggests that this interface is functionally relevant. This work reveals the range of G structural changes and suggests that G monomers can re‐associate, through antiparallel interactions between fusion domains, into dimers that play a role at some early stage of the fusion process.     

Does the combination of citrate and phytase exudation in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> promote the acquisition of endogenous soil organic phosphorus?

Background and Aims

Plant acquisition of endogenous forms of soil phosphorus (P) could reduce external P requirements in agricultural systems. This study investigated the interaction of citrate and phytase exudation in controlling the accumulation of P and depletion of soil organic P by transgenic Nicotiana tabacum plants.

Methods

N. tabacum plant lines including wild-type, vector controls, transgenic plants with single-trait expression of a citrate transporter (A. thaliana frd3) or fungal phytases (phyA: A. niger, P. lycii) and crossed plant lines expressing both traits, were characterized for citrate efflux and phytase exudation. Monocultures and intercropped combinations of single-trait plants were grown in a low available P soil (12 weeks). Plant biomass, shoot P accumulation, rhizosphere soil pH and citrate-extractable-P fractions were determined. Land Equivalent Ratio and complementarity effect was determined in intercropped treatments and multiple-linear-regression was used to predict shoot P accumulation based on plant exudation and soil P depletion.

Results

Crossed plant lines with co-expression of citrate and phytase accumulated more shoot P than single-trait and intercropped plant treatments. Shoot P accumulation was predicted based on phytase-labile soil P, citrate efflux, and phytase activity (Rsq=0.58, P < .0001). Positive complementarity occurred between intercropped citrate- and phytase-exuding plants, with the greatest gains in shoot P occurring in plant treatments with A. niger phyA expression.

Conclusions

We show for the first time that trait synergism associated with the exudation of citrate and phytase by tobacco can be linked to the improved acquisition of P and the depletion of soil organic P.

     

Synthesis of pentacyclic steroids

Due to their high profile biological activity, the steroids are among the most important secondary metabolites. A review of literature on the synthesis of pentacyclic steroids is presented.     

Capacitated acrosome-intact mouse spermatozoa bind to Sepharose beads coated with functional neoglycoproteins

Capacitated acrosome-intact mouse spermatozoa bind to the egg's extracellular coat, the zona pellucida (ZP), in a carbohydrate-mediated receptor-ligand manner. The tight irreversible binding of the opposite gametes triggers a signal transduction pathway resulting in the exocytosis of acrosomal contents (i.e., induction of the acrosome reaction [AR]). Previously, we demonstrated that a hexose (mannose) and two amino sugars (N-acetylglucosamine and N-acetylgalactosamine), when covalently conjugated to bovine serum albumin (BSA) (functional neoglycoproteins, ngps), mimicked mZP3 and induced the AR [Biol. Reprod. 60 (1999) 94-101]. To further elucidate the specificity of sperm-ngp interaction and the mZP3 mimicking role of the functional ngps, we have examined binding of the mouse spermatozoa to Sepharose 4B beads coated with the functional and non-functional ngps as well as BSA, ovalbumin (OVA), or asialofetuin (ASF). A significantly greater number of capacitated acrosome-intact spermatozoa bound to the beads coated with functional ngps than the beads coated with non-functional ngps, BSA, OVA, or ASF. The binding was temperature-sensitive and was highest when the sperm-bead assay was carried out at 37 degrees C. Blocking of in vitro capacitation, by including calmodulin antagonists in the incubation medium, prevented sperm from binding to the beads. Furthermore, inclusion of free sugars (mannose, N-acetylglucosamine, or N-acetylgalactosamine) in the binding assay, either individually or as a mixture, inhibited sperm-bead binding in a concentration-dependent manner. Taken together, our data provide evidence strongly suggesting that binding of capacitated spermatozoa to the ngp-coated Sepharose beads is specific. The beads that mimic zona-intact eggs provide an excellent tool for examining pharmacological effects of reagents that alter the sperm function. In addition, the immobilized ngp(s) will be useful as an affinity medium to isolate the sperm surface receptor(s) that recognize and bind to the sugar residues.     

Nonenzymatic synthesis of glycerolipids catalyzed by imidazole

Imidazole catalyzed acylations of lysolipids by acyl-CoAs in water at room temperature and at a pH close to neutrality. In the presence of oleoyl-CoA and either lysophosphatidylcholine, 1-palmitoyl-sn-glycero-3-phosphocholine (LPC); lysophosphatidylglycerol, monoacyl-sn-glycero-3-phosphoglycerol; lysophosphatidyl acid, 1-oleoyl-sn-glycero-3-phosphate; lysophosphatidylserine, monoacyl-sn-glycero-3-phosphoserin; or lysophosphatidylethanolamine, monoacyl-sn-glycero-3-phosphoethanolamine, the corresponding phospholipids were synthesized. Similarly, the use of lyso-platelet activating factor, an ether analog of LPC, yielded the formation of 1-O-alkyl-2-oleoyl-sn-glycero-3-phosphocholine. In the presence of LPC, an imidazole-catalyzed synthesis of phosphatidylcholine (PC) occurred when medium, long, and very long chain acyl-CoAs were added. With hydroxyacyl-CoA, a similar PC synthesis was obtained. The process described in the present paper appears to offer several potential applications of interest for the synthesis of glycerophospholipids and triglycerides with labeled and/or an unusual or fragile fatty acid, or when suitable acyltransferases have not yet been described in the literature and/or are not commercially available. The method described is very safe and simple since lipids can be synthesized in tubes containing 0.7% imidazole in water, and left for a few hours at room temperature on the bench.     

A positive feedback loop stabilizes the guanine-nucleotide exchange factor Cdc24 at sites of polarization

In Saccharomyces cerevisiae, activation of Cdc42 by its guanine-nucleotide exchange factor Cdc24 triggers polarization of the actin cytoskeleton at bud emergence and in response to mating pheromones. The adaptor protein Bem1 localizes to sites of polarized growth where it interacts with Cdc42, Cdc24 and the PAK-like kinase Cla4. We have isolated Bem1 mutants (Bem1-m), which are specifically defective for binding to Cdc24. The mutations map within the conserved PB1 domain, which is necessary and sufficient to interact with the octicos peptide repeat (OPR) motif of Cdc24. Although Bem1-m mutant proteins localize normally, bem1-m cells are unable to maintain Cdc24 at sites of polarized growth. As a consequence, they are defective for apical bud growth and the formation of mating projections. Localization of Bem1 to the incipient bud site requires activated Cdc42, and conversely, expression of Cdc42-GTP is sufficient to accumulate Bem1 at the plasma membrane. Thus, our results suggest that Bem1 functions in a positive feedback loop: local activation of Cdc24 produces Cdc42-GTP, which recruits Bem1. In turn, Bem1 stabilizes Cdc24 at the site of polarization, leading to apical growth.     

Cell cycle-dependent nuclear export of Cdh1p may contribute to the inactivation of APC/C(Cdh1)

Cdh1p is a substrate-specific subunit of the anaphase-promoting complex (APC/C), which functions as an E3 ubiquitin ligase to degrade the mitotic cyclin Clb2p and other substrates during the G(1) phase of the cell cycle. Cdh1p is phosphorylated and thereby inactivated at the G(1)/S transition predominantly by Cdc28p-Clb5p. Here we show that Cdh1p is nuclear during the G(1) phase of the cell cycle, but redistributes to the cytoplasm between S phase and the end of mitosis. Nuclear export of Cdh1p is regulated by phosphorylation and requires active Cdc28p kinase. Cdh1p binds to the importin Pse1p and the exportin Msn5p, which is necessary and sufficient to promote efficient export of Cdh1p in vivo. Although msn5delta cells are viable, they are sensitive to Cdh1p overexpression. Likewise, a mutant form of Cdh1p, which is constitutively nuclear, prevents accumulation of Clb2p and leads to cell cycle arrest when overexpressed in wild-type cells. Taken together, these results suggest that phosphorylation-dependent nuclear export of Cdh1p by Msn5p contributes to efficient inactivation of APC/C(Cdh1).     

Inhibition of the plastidial phosphatidylcholine synthesis by silver, copper, lead and mercury induced by formation of mercaptides with the lyso-PC acyltransferase

Plastids greatly rely on the import of extraplastidial precursors for the synthesis of their own lipids, and several studies have shown that a lyso-PC acyltransferase located in the envelope may be involved in the import process. Because the presence of heavy metals in soil or in nutrient solutions induces changes in the lipid composition of plastid membranes (and therefore greatly reduces the photosynthetic capability of plants), we analysed the effect of several metal salts on plastidial lyso-PC acyltransferase activity. Among the 12 heavy metals studied, silver, copper, mercury and lead inhibited this activity. Metal bound to the enzyme was not - or only very slightly - released from the protein except when thiol-reducing agents (and not imidazole) were added. The results strongly suggest that the inhibitory effect is due to a formation of mercaptide between metal and cysteine(s). The relationship between the inhibition of the plastidial lyso-PC acyltransferase activity and the in vivo effects of metal salts on the plastid membranes is discussed.     

Bystander cell killing spreading from endothelial to tumor cells in a three-dimensional multicellular nodule model after Escherichia coli nitroreductase gene delivery

Tumor cells are elusive targets for standard anticancer chemotherapy due to their heterogeneity and genetic instability. On the other hand, proliferating host endothelial cells (ECs) are genetically stable and have a low mutational rate. Thus, antiangiogenic therapy directed against tumor's ECs should, in principle, improve the efficacy of antitumor therapy by inducing little or no drug resistance. Here we present a gene-directed enzyme prodrug therapy (GDEPT) strategy for targeting the tumor vasculature, using the Escherichia coli nitroreductase (ntr) gene delivery associated with the treatment with the prodrug CB1954. In a first time we demonstrated the ability of the ntr/CB1954 system to induce an apoptotic-mediated cell death on monolayer cultures of human umbilical vein ECs (HUV-EC-C). Then, when ntr-transfected HUV-EC-C cells (HUV-EC-C/ntr(+)) were associated in a three-dimensional (3-D) multicellular nodule model with untransfected B16-F10 murine melanoma cell line, we observed a CB1954-mediated bystander cell killing effect from endothelial to neighboring melanoma cells. To our knowledge, this is the first report indicating that GDEPT-based antiangiogenic targeting may be an effective approach for cancer treatment relied on the spreading of the bystander effect from endothelial to tumor cells.     

pKa and volume of residue one influence delta/mu opioid binding: QSAR analysis of tyrosine replacement in a nonselective deltorphin analogue

[Gly(4)]deltorphin (Tyr-D-Ala-Phe-Gly-Val-Val-Gly-NH(2)) is a nonselective analogue of the opioid heptapeptides isolated from Phyllomedusa amphibian skin. Its nonselective nature allows for simultaneous characterization of the effects of sequence modification on both delta (delta) and mu (mu) receptor binding. The N-terminal regions of opioid peptides are considered to be responsible for receptor recognition, and the tyrosine at position one is relatively intolerant to alteration. In order to further investigate the role of the phenolic hydroxyl group in receptor interaction, a series of peptides was synthesized in which the position-one tyrosine residue was replaced with analogues of varying electronic, steric, and acid/base character, including ring-substituted tyrosines, para-substituted phenylalanines, and other nonaromatic and heterocyclic amino acids. The effects of these replacements on delta and mu receptor affinities were measured and then analyzed through quantitative structure-activity relationship (QSAR) calculations. Results support a dual hydrogen bond donor/acceptor role for the Tyr(1) hydroxyl moiety, with less acidic hydroxyl groups exhibiting stronger binding to opioid receptors. In addition, steric bulk in the Tyr(1) position independently strengthens mu and possibly delta binding, presumably by either a ligand conformational effect or enhanced van der Waals interactions with a 'loose' receptor site. The pK(a) effect is stronger on delta than on mu binding, generating an increase in delta selectivity with increasing residue-one pK(a).