Biotinylated Cyclooligosaccharides for Paclitaxel Solubilization.

The poor water solubility of paclitaxel causes significant problems in producing cancer therapeutic formulations. Here, we aimed to solubilize paclitaxel using biocompatible cyclic carbohydrates. Generally recognized as safe, labeled ß-cyclodextrin (ß-CD), a cyclic α-1,4-glucan consisting of seven glucoses, was prepared, and bio-sourced cyclosophoraoses (CyS), which are unbranched cyclic ß-1,2-glucans with 17-23 glucose units, were purified using various chromatographic methods from Rhizobium leguminosarum cultural broth. For effective targeting, CyS and ß-CD were modified with a biotinyl moiety in a reaction of mono-6-amino CyS and mono-6-amino-ß-CD with N-hydroxysuccinimide ester of biotinamidohexanoic acid. Interestingly, the aqueous solubility of paclitaxel was enhanced 10.3- and 3.7-fold in the presence of biotinyl CyS and biotinyl ß-CD, respectively. These findings suggest that biotin-appended cyclooligosaccharides can be applied to improve the delivery of paclitaxel.

Rhizobium leguminosarum HupE is a highly-specific diffusion facilitator for nickel uptake.

Bacteria require nickel transporters for the synthesis of Ni-containing metalloenzymes in natural, low nickel habitats. In this work we carry out functional and topological characterization of Rhizobium leguminosarum HupE, a nickel permease required for the provision of this element for [NiFe] hydrogenase synthesis. Expression studies in the Escherichia coli nikABCDE mutant strain HYD723 revealed that HupE is a medium-affinity permease (apparent Km 227 ± 21 nM; Vmax 49 ± 21 pmol Ni(2+) min(-1) mg(-1) bacterial dry weight) that functions as an energy-independent diffusion facilitator for the uptake of Ni(ii) ions. This Ni(2+) transport is not inhibited by similar cations such as Mn(2+), Zn(2+), or Co(2+), but is blocked by Cu(2+). Analysis of site-directed HupE mutants allowed the identification of several residues (H36, D42, H43, F69, E90, H130, and E133) that are essential for HupE-mediated Ni uptake in E. coli cells. By using translational fusions to reporter genes we demonstrated the presence of five transmembrane domains with a periplasmic N-terminal domain and a C-terminal domain buried in the lipid bilayer. The periplasmic N-terminal domain contributes to stability and functionality of the protein.

Computational study of the Fe(CN)2CO cofactor and its binding to HypC protein.

In the intricate maturation process of [NiFe]-hydrogenases, the Fe(CN)2CO cofactor is first assembled in a HypCD complex with iron coordinated by cysteines from both proteins and CO is added after ligation of cyanides. The small accessory protein HypC is known to play a role in delivering the cofactor needed for assembling the hydrogenase active site. However, the chemical nature of the Fe(CN)2CO moiety and the stability of the cofactor-HypC complex are open questions. In this work, we address geometries, properties, and the nature of bonding of all chemical species involved in formation and binding of the cofactor by means of quantum calculations. We also study the influence of environmental effects and binding to cysteines on vibrational frequencies of stretching modes of CO and CN used to detect the presence of Fe(CN)2CO. Carbon monoxide is found to be much more sensitive to sulfur binding and the polarity of the medium than cyanides. The stability of the HypC-cofactor complex is analyzed by means of molecular dynamics simulation of cofactor-free and cofactor-bound forms of HypC. The results show that HypC is stable enough to carry the cofactor, but since its binding cysteine is located at the N-terminal unstructured tail, it presents large motions in solution, which suggests the need for a guiding interaction to achieve delivery of the cofactor.

Efectividad de cepas rizobianas de frijolbajo diferentes regímenes de fósforo / Effectiveness of cowpea rhizobial strains under different phosphorus regimes

En Venezuela, el frijol representa una alternativa a la proteína animal, debido a su alto consumo y valor nutritivo, por ello se ha estimulado la implementación de programas para reactivar la economía de los pequeños y medianos productores, a fin de incrementar su producción y así tener mayor disponibilidad de proteína de alta calidad a bajo costo; de manera que, los estudios encaminados a mejorar su cultivo, son acertados. Se evaluó la efectividad de cepas rizobianas de crecimiento lento (cl) y rápido (cr) en frijol (Vigna unguiculata (L.) Walp.) cultivar TC9-6 en varios regímenes de fósforo (0, 20, 40 y 80 kgP2O5 ha-1), con un diseño experimental de bloques al azar con arreglo factorial. Las plantas se cultivaron en 4 kg de suelo de sabana 45 días y las cepas en caldo de levadura y manitol: 5 (cr: JV91) y 10 (cl: JV94) días. La inoculación (2 ml cada vez) fue aplicada a la siembra y 6 días más tarde. La utilización de fósforo (40-80 kgP2O5 ha-1) incrementó la nodulación (número, peso seco total e individual de nódulos) y favoreció la aparición de nódulos rojos; así mismo, acrecentó el peso de la materia seca, la altura, el número de hojas y la concentración de nitrógeno del vástago. Los valores fueron similares con ambos tipos de cepas (efectividad similar) y para las dos concentraciones (40-80 kgP2O5 ha-1), con las menores estimaciones para 0 y 20 kgP2O5 ha-1. De acuerdo con los resultados las concentraciones de 40 y 80 kgP2O5 ha-1 fueron las más favorables para el crecimiento y la nodulación de frijol.

In Venezuela, cowpea is an alternative to animal protein due to its high consumption and nutritious value, so it has stimulated the implementation of programs to reactivate the small and medium producers economy, in order to increase its production and to have major high quality protein availability at low cost; so that, the studies carry on to improve its cultivation, are well-aimed. The effectiveness of slow (sg) and fast (fg) growing rhizobial strains was evaluated in cowpea (Vigna unguiculata (L.) Walp) cultivar TC9-6 at various phosphorus regimes (0, 20, 40 and 80 kgP2O5 ha-1): randomized block design with factorial arrangement. Plants were cultivated in 4 kg savannah soil: 45 days, and the strains in yeast and mannitol broth: 5 (fg: JV91) and 10 (sg: JV94) days. The inoculation (2 ml each time) was applied at sowing time and 6 days later. Phosphorus utilization (40-80 kgP2O5 ha-1) increased nodulation (nodule number, total and individual dry weight) and favoured nodule red colour appearance; also, incremented shoot dry matter weight, height, leaves number and nitrogen concentration. Values were similar with both strain types (similar effectiveness) and to the two doses (40-80 kgP2O5 ha-1), with lower estimations to 0 and 20 kgP2O5 ha-1. Accordingly with the results, the doses of 40 and 80 kgP2O5 ha-1 were the most favourable to cowpea growth and nodulation.

Functional identification of ATP-driven Ca2+ pump in the peribacteroid membrane of broad bean root nodules.

A Ca2+ indicator arsenazo III was used to demonstrate calcium uptake activity of symbiosomes and the peribacteroid membrane (PBM) vesicles isolated from broad bean root nodules and placed in the medium containing ATP and Mg2+ ions. This process was shown to be rapidly stopped by vanadate, completely reversed in the presence of the calcium ionophore A23187 but insensitive to agents abolishing electrical potential or pH difference across the PBM. The presence of an endogenous calcium pool within isolated symbiosomes and bacteroids was detected using a Ca2+ indicator chlortetracycline. These results prove a primary active transport of Ca2+ through the PBM of legume root nodules and provide the first functional identification of an ATP-driven Ca2+-pump, most likely Mg2+-dependent Ca2+-translocating ATPase, in this membrane.

Purification of pea nodule symbiosomes using an aqueous polymer two-phase system.

Symbiosomes were obtained from mature pea (Pisum sativum cv. Argona) root nodules infected with Rhizobium leguminosarum strain (biov. viciae 3841) and purified using an aqueous polymer two-phase system (APS). The APS consists of a mixture of polymers, usually dextran T500 and poly(ethylene glycol) 3350, prepared as aqueous solutions on a weight per weight basis, where each fraction distributes according to their surface characteristics. Results of ATPase activity, cytochrome c oxidase activity, glucan synthase II activity, NAD(P)H-cytochrome c reductase activity, NO3(-)-sensitive ATPase activity, transport of [14C]malate vs. [14C]glutamate and MAC 57 antigen analysis showed that the APS method provided intact symbiosomes with low bacteroid, plasma membrane, endoplasmic reticulum and/or mitochondria contamination. No complicated equipment is needed and the method was simple and fast, compared with other purification techniques.

Photosynthetic symbionts of Aeschynomene spp. form a cluster with bradyrhizobia on the basis of fatty acid and rRNA analyses.

The relationship between photosynthetic rhizobia that nodulate 10 Aeschynomene species (Aeschynomene afraspera, Aeschynomene denticulata, Aeschynomene evenia, Aeschynomene indica, Aeschynomene nilotica, Aeschynomene pratensis, Aeschynomene rudis, Aeschynomene scabra, Aeschynomene schimperi, and Aeschynomene sensitiva) and reference strains of the genera Bradyrhizobium, Rhizobium, and Azorhizobium was investigated by analyzing cellular fatty acid methyl esters (FAME) and 16S rRNA sequences. The members of each genus produced very distinct FAME patterns, and the photosynthetic rhizobia formed a subcluster in the Bradyrhizobium cluster. The absence of the cyc C19:0 type of fatty acid in all of the photosynthetic rhizobium strains isolated from 10 Aeschynomene species distinguished these microorganisms from other known rhizobia, including strain BTAi 1, a photosynthetic symbiont of A. indica. We sequenced a 264-base segment of the 16S rRNA genes of selected strains after amplification by the PCR and compared the results with previously published sequences for species of rhizobia and related photosynthetic bacteria. Photosynthetic strains IRBG 2 (from A. afraspera), IRBG 230 (from A. nilotica), and ORS 322 (from A. afraspera) had identical sequences but were distinct from strain BTAi (from A. indica) and from strain IRBG 231 (from A. denticulata), which is similar to the type strain (DNA homology group Ia) of Bradyrhizobium japonicum. Nonphotosynthetic strain IRBG 274 (from A. afraspera) was closely related to Bradyrhizobium elkanii (DNA homology group II). All of the photosynthetic rhizobia clearly fell into the Bradyrhizobium cluster. Although the results of the FAME and 16S rRNA analyses were in excellent agreement, our placement of the photosynthetic rhizobia is in apparent conflict with phenotypic data, as determined by numerical taxonomy (Ladha and So, Int. J. Syst. Bacteriol., in press) which placed the photosynthetic rhizobia in a coherent cluster that is as far from the genus Bradyrhizobium as the genera Rhizobium and Azorhizobium are. While the FAME and 16S rRNA data probably provide a more reliable indication of phylogeny, the degree of phenotypic divergence observed raises questions concerning the polyphasic approach to bacterial systematics.

Composiçäo química e valor nutricional de cultivares de gräo-de-bico produzidos no Estado de Säo Paulo / Chemical composition and nutritional value of chickpea cultivars grown in the State of Säo Paulo

Foram analisados a composiçäo química e valor nutricional de cultivares de gräo-de-bico produzidos no Estado de Säo Paulo. Estudou-se também seu aproveitamento na forma de farinha. O cultivar IAC-México apresentou maior teor de proteína (19,7 por cento). O teor de fibra alimentar total foi maior para as sementes marrom e preta. Todos os cultivares apresentaram menor atividade de inibidor de tripsina que a soja e as sementes escuras apresentaram teores maiores de compostos fenólicos do que as sementes claras. A composiçäo em minerais demonstra que este gräo é uma boa fonte de fósforo, magnésio, ferro, potássio e manganês. Embora deficientes em aminoácidos sulfurados, os cultivares apresentaram um quociente de eficiência protéica líquida (NPR) e quociente de eficiência protéica (PER) superiores a 80 por cento do valor obtido para a caseína e digestibilidade em torno dos 80 por cento

Composiçäo química, processamento e avaliaçäo do broto-de-bambu D. giganteus Munro processado / Chemical composition, processing and evaluation of bamboo shoots D. giganteus Munro

O broto-de-bambu Dendrocalamus giganteus Munro, dividido nas porçöes apical e basal, foi estudado quanto aos aspectos de composiçäo química, processamento e características físicas e organolépticas de aceitabilidade do produto processado. As etapas envolvidas no processamento foram discutidas detalhadamente. O broto-de-bambu se caracteriza como vegetal de baixa acidez e apresenta níveis de HCN no produto processado foram da ordem de 742 mg/kg e 532 mg/kg nas porçöes apical e basal, respectivamente. Os níveis máximos residuais de HCN no produto processado foram da ordem de 76,0 e 59,4 mg/kg para as porçöes apical e basal. O rendimento obtido de broto-de-bambu processado foi de 35,9 por cento em relaçäo à matéria-prima. As duas porçöes do broto-de-bambu foram classificadas entre o "gostei" e "gostei muito" da escala hedônica facial empregada no teste de aceitaçäo