Structure determination of a tetrahydro-beta-carboline of arthropod origin: a novel alkaloid-toxin subclass from the web of spider Nephila clavipes

The orb-web spiders are polyphagous animals in which the web plays a very important role in the capture of preys; oily droplets usually cover the capture-web of the spider Nephila clavipes and seem to be of great importance for prey capture. The knowledge of the chemical composition of these droplets is necessary to understand the function of this adhesive material in web mechanics and prey capture. A novel subclass of spider toxins, tetrahydro-beta-carboline, was identified among the weaponry of compounds present inside of oily droplets. This type of alkaloid is not common among the natural compounds of spider toxins. Apparently, when the prey arthropods get caught by the spider web, their bodies are covered with many adhesive oily droplets, which disrupt delivering the tetrahydro-beta-carboline to the direct contact with the prey integument. Toxicity assays demonstrated a potent lethal effect of the alkaloid toxin to the spider preys; topical applications of the tetrahydro-beta-carboline at first caused clear signs of neurotoxicity, followed by the death of preys. The structure of the major component, a tetrahydro-beta-carboline, among the alkaloid toxins was elucidated by means of UV spectrophotometry, ESI mass spectrometry, 1H-NMR spectroscopy, and high-resolution mass spectrometry. The structure of the natural toxin was determined as 1-(2-guanidinoethyl)-1,2,3,4-tetrahydro-6-hydroxymethyl)-beta-carboline; the investigation of the pharmacological properties and neurotoxic actions of this compound may be used in the future as reference for the development of new drugs to be applied at level of pest control in agriculture.     

Transgene elimination in genetically modified dry bean and soybean lines

Transgene elimination is a poorly studied phenomenon in plants. We made genetic and molecular studies of a transgenic dry bean line immune to bean golden mosaic geminivirus and a soybean line. In both lines, the transgenes were stable during the vegetative phase but were eliminated during meiosis. Due to its potential biotechnological value, this transgenic line was micropropagated by grafting and the vegetative copies were studied for more than two years. More than 300 plants of progeny were obtained during this period, demonstrating that the phenomenon of elimination was consistently repeated and offering an opportunity for detailed study of transgene elimination, including the characterization of the integration sites. Cloning and sequencing of the transgenic loci, reciprocal crosses to untransformed plants, genomic DNA blots, and GUS assays were performed in the transgenic lines. Based on the molecular and genetic characterization, possible mechanisms involved in transgene elimination include intrachromosomal recombination, genetic instability resulting from the tissue culture manipulations, and co-elimination of transgenes, triggered by a process of genome defense.     

Functional analysis of two divalent metal-dependent regulatory genes dmdR1 and dmdR2 in Streptomyces coelicolor and proteome changes in deletion mutants

In Gram-positive bacteria, the expression of iron-regulated genes is mediated by a class of divalent metal-dependent regulatory (DmdR) proteins. We cloned and characterized two dmdR genes of Streptomyces coelicolor that were located in two different nonoverlapping cosmids. Functional analysis of dmdR1 and dmdR2 was performed by deletion of each copy. Deletion of dmdR1 resulted in the derepression of at least eight proteins and in the repression of three others, as shown by 2D proteome analysis. These 11 proteins were characterized by MALDI-TOF peptide mass fingerprinting. The proteins that show an increased level in the mutant correspond to a DNA-binding hemoprotein, iron-metabolism proteins and several divalent metal-regulated enzymes. The levels of two other proteins--a superoxide dismutase and a specific glutamatic dehydrogenase--were found to decrease in this mutant. Complementation of the dmdR1-deletion mutant with the wild-type dmdR1 allele restored the normal proteome profile. By contrast, deletion of dmdR2 did not affect significantly the protein profile of S. coelicolor. One of the proteins (P1, a phosphatidylethanolamine-binding protein), overexpressed in the dmdR1-deleted mutant, is encoded by ORF3 located immediately upstream of dmdR2; expression of both ORF3 and dmdR2 is negatively controlled by DmdR1. Western blot analysis confirmed that dmdR2 is only expressed when dmdR1 is disrupted. Species of Streptomyces have evolved an elaborated regulatory mechanism mediated by the DmdR proteins to control the expression of divalent metal-regulated genes.     

Azo dye reduction by mesophilic and thermophilic anaerobic consortia

The reduction of the azo dye model compounds Reactive Red 2 (RR2) and Reactive Orange 14 (RO14) by mesophilic (30 degrees C) and thermophilic (55 degrees C) anaerobic consortia was studied in batch assays. The contribution of fermentative and methanogenic microorganisms in both temperatures was evaluated in the presence of the fermentative substrate glucose and the methanogenic substrates acetate, H2/CO2, methanol, and formate. Additionally, the effect of the redox mediator riboflavin on electron shuttling was assessed. We concluded that the application of thermophilic anaerobic treatment is an interesting option for the reductive decolorization of azo dyes compared to mesophilic conditions. The use of high temperature may decrease or even take the place of the need for continuous redox mediator dosage in bioreactors, contrarily to the evident effect of those compounds on dye reduction under mesophilic conditions. Both fermenters and methanogens may play an important role during reductive decolorization of dyes, in which mediators are important not only for allowing the different microbes to participate more effectively in this complex reductive biochemistry but also for assisting in the competition for electrons between dyes and other organic and inorganic electron acceptors.     

Enhanced production of hydroperoxides by immobilized lipoxygenase in the presence of cyclodextrins

The advantages of the presence of cyclodextrins in a reaction catalyzed by immobilized lipoxygenase at neutral pH are reported for the first time. The steady-state rate in the presence of beta-cyclodextrins was seven times higher than in control experiments using the same concentration of linoleic acid; furthermore the percentage of substrate conversion (and product accumulation) obtained in the presence of beta-cyclodextrins was higher than in the control assays. The optimum concentration of free linoleic acid coincided with the critical micellar concentration for linoleic acid at neutral pH. The operational stability of the immobilized enzyme increased in the presence of beta-cyclodextins, while an increase in the percentage of 13-HPOD was also observed.     

An approximate solution for a transient two-phase stirred tank bioreactor with nonlinear kinetics

The derivation of an approximate solution method for models of a continuous stirred tank bioreactor where the reaction takes place in pellets suspended in a well-mixed fluid is presented. It is assumed that the reaction follows a Michaelis-Menten-type kinetics. Analytic solution of the differential equations is obtained by expanding the reaction rate expression at pellet surface concentration using Taylor series. The concept of a pellet's dead zone is incorporated; improving the predictions and avoiding negative values of the reagent concentration. The results include the concentration expressions obtained for (a) the steady state, (b) the transient case, imposing the quasi-steady-state assumption for the pellet equation, and (c) the complete solution of the approximate transient problem. The convenience of the approximate method is assessed by comparison of the predictions with the ones obtained from the numerical solution of the original problem. The differences are in general quite acceptable.     

Structures of the iron-sulfur flavoproteins from Methanosarcina thermophila and Archaeoglobus fulgidus

Iron-sulfur flavoproteins (ISF) constitute a widespread family of redox-active proteins in anaerobic prokaryotes. Based on sequence homologies, their overall structure is expected to be similar to that of flavodoxins, but in addition to a flavin mononucleotide cofactor they also contain a cubane-type [4Fe:4S] cluster. In order to gain further insight into the function and properties of ISF, the three-dimensional structures of two ISF homologs, one from the thermophilic methanogen Methanosarcina thermophila and one from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus, were determined. The structures indicate that ISF assembles to form a tetramer and that electron transfer between the two types of redox cofactors requires oligomerization to juxtapose the flavin mononucleotide and [4Fe:4S] cluster bound to different subunits. This is only possible between different monomers upon oligomerization. Fundamental differences in the surface properties of the two ISF homologs underscore the diversity encountered within this protein family.     

Macromolecular accessibility of fluorescent taxoids bound at a paclitaxel binding site in the microtubule surface

The macromolecular accessibility of the paclitaxel binding site in microtubules has been investigated using a fluorescent taxoid and antibodies against fluorescein, which cannot diffuse into the microtubule lumen. The formation of a specific ternary complex of microtubules, Hexaflutax (7-O-{N-[6-(fluorescein-4'-carboxamido)-n-hexanoyl]-l-alanyl}paclitaxel) and 4-4-20 IgG (a monoclonal antibody against fluorescein) has been observed by means of sedimentation and electron microscopy methods. The kinetics of binding of the antibody to microtubule-bound Hexaflutax has been measured. The quenching of the observed fluorescence is fast (k+ 2.26 +/- 0.25 x 10(6) m(-1) s(-1) at 37 degrees C), indicating that the fluorescein groups of Hexaflutax are exposed to the outer solvent. The velocity of the reaction is linearly dependent on the antibody concentration, indicating that a bimolecular reaction is being observed. Another fluorescent taxoid (Flutax-2) bound to microtubules has also been shown to be rapidly accessible to polyclonal antibodies directed against fluorescein. A reduced rate of Hexaflutax quenching by the antibody is observed in microtubule-associated proteins containing microtubules or in native cellular cytoskeletons. It can be concluded that the fluorescent taxoids bind to an outer site on the microtubules that is shared with paclitaxel. Paclitaxel would be internalized in a further step of binding to reach the known luminal site, this step being blocked in the case of the fluorescent taxoids. Because the fluorescent ligands are able to induce microtubule assembly, binding to the outer site should be enough to induce assembly by a preferential binding mechanism.     

Structural dissection of a highly knotted peptide reveals minimal motif with antimicrobial activity

The increasing occurrence of bacterial resistance to antibiotics is driving a renewed interest on antimicrobial peptides, in the hope that understanding the structural features responsible for their activity will provide leads into new anti-infective drug candidates. Most chemical studies in this field have focused on linear peptides of various eukaryotic origins, rather than on structures with complex folding patterns found also in nature. We have undertaken the structural dissection of a highly knotted, cysteine-rich plant thionin, with the aim of defining a minimal, synthetically accessible, structure that preserves the bioactive properties of the parent peptide. Using efficient strategies for directed disulfide bond formation, we have prepared a substantially simplified (45% size reduction) version with undiminished antimicrobial activity against a representative panel of pathogens. Analysis by circular dichroism shows that the downsized peptide preserves the central double alpha-helix of the parent form as an essential bioactive motif. Membrane permeability and surface plasmon resonance studies confirm that the mechanism of action remains unchanged.     

Epithelium and/or theca are required for ATP-elicited K+ current in follicle-enclosed Xenopus oocytes

The Xenopus follicular cell membrane is endowed with ATP-sensitive K+ channels, which are operated by various transmitters. These generate the ionic response named IK,cAMP via a mechanism that involves intracellular cAMP synthesis. It is known that opening these K+ channels favors oocyte maturation. Follicle stimulation by adenosine (Ado) or ATP consistently generates a strong IK,cAMP via activation of P1 and P3 purinergic receptors; however, ATP can also inhibit IK,cAMP, apparently acting on a third receptor type. Here, we show that IK,cAMP might be elicited by ATP released within the follicle, and that current activation by ATP was entirely dependent on the presence of epithelial and/or theca layers. Morphological studies confirmed that removal of epithelium/theca in these follicles (e.t.r.) was complete, and activation of fast Cl- (Fin) currents by ATP in e.t.r. follicles confirmed that communication between oocyte and follicular cells remained unchanged. Thus, dependence on epithelium/theca was specific for ATP-elicited K+ current. Using UTP and betagamma-MeATP as specific purinergic agents for IK,cAMP inhibition and activation, respectively, it was found that inhibition of IK,cAMP elicited by ATP or UTP was robustly present in e.t.r. follicles but was absent or strongly decreased in whole follicles (w.f.). Accordingly, this indicated that in w.f., epithelium and/or theca downregulated the IK,cAMP inhibition evoked by ATP, and that this control mechanism was absent in e.t.r. follicles. We suggest that this notable action on follicular cells involves one or both of two mechanisms, a paracrine transmitter released from epithelial and/or theca layers and action of ecto-ATPases.