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.     

Movement, toxicity, and persistence of imidacloprid in seedling Tabasco pepper infested with Myzus persicae (Hemiptera: Aphididae)

Application of imidacloprid to the soil in which Tabasco pepper, Capsicum frutescens L., seedlings were growing was highly effective against the green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae). In just 48 h after the soil drench, aphid numbers on treated plants declined from 292.1 to 33.0 per plant, a reduction of 89%. By 72 and 96 h after the application, the reductions were 97 and 100%, respectively. Reductions in green peach aphid numbers also indicated that imidacloprid readily moved throughout the Tabasco pepper plant. Although, initial green peach aphid reductions at 24 and 48 h after imidacloprid application to soil, were greater on the lower leaves than on the upper leaves, by 72 h toxicity was high throughout the plant. At 48 h, overall green peach aphid reduction on seedlings grown in wet soil was significantly higher than that on plants growing in the drier soil. Regardless of soil moisture or leaf location, no live green peach aphids were detected on treated seedlings after 96 h. After the initial uptake period, toxicity to green peach aphid remained high for 5 wk. Under Tabasco pepper production conditions in Central America, the greatest need for aphid management is just after transplanting. Imidacloprid soil drenches before transplanting should offer the Tabasco pepper producer an extended period of aphid-free production.     

Reproductive pattern in agrarian and immigrant receptor populations: a survey of El Ejido (SE Spain)

This paper deals with the fertility pattern of the El Ejido population, an agricultural Spanish community characterised by the rapid development of its modern agrarian economy. Consequently, the arrival of immigrants has sharply increased throughout the second half of the twentieth century, with important demographic consequences as well as reproductive changes. The analyses of the age-specific fertility rate (fx) and the total fertility rate (TFR) were used in order to describe the reproductive pattern of this population in 2000. The main characteristics were the following: a) Regarding the temporal change, an important decrease of fx has been observed in all age groups for the last twenty years, as a consequence of progressive birth control. However, the reproductive pattern has kept almost invariable and has been characterised by a maximum fertility at age group of 25-29 years old. b) Regarding the general Spanish fertility, the comparison of fx in both populations suggests a younger maternity in the agricultural population than in the national, the maximum fertility delayed to the 30-34 age group. c) Moreover, the El Ejido population showed a clear higher offspring per woman (TFR = 1.42) than the national (TFR = 1.24), according to the agrarian character of the El Ejido population. d) Finally, this greater reproductive level of El Ejido is also due to the arrival of women at fertile age, who come mainly from Africa, and above all from Morocco.     

Low tumor cell density environment yields survival advantage of tumor cells exposed to MTX in vitro

Stable resistance to methotrexate has been well characterized after prolonged treatment of the HT-29 colon cancer cell line, but the mechanism of cell survival at the early stages of the drug resistance process still remains unclear. Here, we demonstrate that human cancer cells in vitro are sensitive to methotrexate only above a critical cell culture density, which specifically coincides with their ability to deplete the extracellular nucleosides from a fully supplemented culture medium. At lower cell densities, extracellular nucleosides remain intact and allow salvage nucleotide synthesis that renders cells insensitive to the drug. Consistently, medium conditioned by cells seeded at standard cell densities sensitizes low cell density cultures. Extracellular nucleosides are the determinants of sensitivity because the latter effect can be mimicked with the use of inhibitors of nucleoside cellular import and reversed by supplying exogenous thymidine and hypoxanthine. Interestingly, treatment at a sensitizing cell density does not preclude the survival of less than 1% of the cells--which have no intrinsic resistance--owing to the inability of the dying cell population to condition the culture medium; this population thus survives indefinitely to continuous treatment by keeping adapted to a low cell number. This cell density-dependent adaptive process accounts for the initial steps of in vitro resistance to methotrexate (MTX) and provides a novel mechanistic insight into the cell population dynamics of cell survival and cell death during drug treatment.