Effect of substituent on the thermodynamics of D-glucopyranoside binding to concanavalin A, pea (Pisum sativum) lectin and lentil (Lens culinaris) lectin

Titration calorimetry measurements of the binding of phenyl-alpha (alpha PhOGlu), 3-methoxy (3MeOGlu), fluorodeoxy and deoxy derivatives of alpha-D-glucopyranose (Glu) to concanavalin A (conA), pea lectin and lentil lectin were performed at approx. 10 and 25 degrees C in 0.01 M dimethylglutaric acid/NaOH buffer, pH 6.9, containing 0.15 M NaCl and Mn2+ and Ca2+ ions. Apparently the 3-deoxy, 4-deoxy and 6-deoxy as well as the 4-fluorodeoxy and 6-fluorodeoxy derivatives of Glu do not bind to the lectins because no heat release was observed on the addition of aliquots of solutions of these derivatives to the lectin solutions. The binding enthalpies, delta H0b, and entropies, delta S0b, determined from the measurements were compared with the same thermodynamic binding parameters for Glu, D-mannopyranoside and methyl-alpha- D-glucopyranoside (alpha MeOGlu). The binding reactions are enthalpically driven with little change in the heat capacity on binding, and exhibit enthalpy-entropy compensation. Differences between the thermodynamic binding parameters can be rationalized in terms of the interactions apparent in the known crystal structures of the methyl-alpha-D-mannopyranoside-conA [Derewenda, Yariv, Helliwell, Kalb (Gilboa), Dodson, Papiz, Wan and Campbell (1989) EMBO J. 8, 2189-2193] and pea lectin-trimanno-pyranoside [Rini, Hardman, Einspahr, Suddath and Carber (1993) J. Biol. Chem. 268, 10126-10132] complexes. Increases in the entropy change on binding are observed for alpha MeOGlu binding to pea and lentil lectin, for alpha PhOGlu binding to conA and pea lectin, and for 3MeOGlu binding to pea lectin relative to the entropy change for Glu binding, and imply that the phenoxy and methoxy substituents provide additional hydrophobic interactions in the complex. Increases in the binding enthalpy relative to that of Glu are observed for deoxy and fluoro derivatives in the C-1 and C-2 positions and imply that these substituents weaken the interaction with the surrounding water, thereby strengthening the interaction with the binding site.

Thermodynamics of monosaccharide binding to concanavalin A, pea (Pisum sativum) lectin, and lentil (Lens culinaris) lectin

Titration calorimetry measurements of the binding of methyl alpha-D-mannopyranoside (Me alpha Man), D-mannopyranoside (Man), methyl alpha-D-glucopyranoside (Me alpha Glu), and D-glucopyranoside (Glu) to concanavalin A (Con A), pea lectin, and lentil lectin were performed at 281 and 292 K in 0.01 M dimethylglutaric acid-NaOH buffer (pH 6.9) containing 0.15 M NaCl and Mn+2 and Ca+2 ions. The site binding enthalpies, delta H, are the same at both temperatures and range from -28.4 +/- 0.9 (Me alpha Man) to -16.6 +/- 0.5 kJ mol-1 (Glu) for Con A, from -26.2 +/- 1.1 (Me alpha Man) to -12.8 +/- 0.4 kJ mol-1 (Me alpha Glu) for pea lectin, and from -16.6 +/- 0.7 (Me alpha Man) to -8.0 +/- 0.2 kJ mol-1 (Me alpha Glu) for lentil lectin. The site binding constants range from 17 +/- 1 x 10(3) M-1 (Me alpha Man to Con A at 281.2 K) to 230 +/- 20 M-1 (Glu to lentil lectin at 292.6 K) and exhibit high specificity for Con A where they are in the Me alpha Man:Man:Me alpha Glu:Glu ratio of 21:4:5:1, while the corresponding ratio is 5:2:1.5:1 for pea lectin and 4:2:2:1 for lentil lectin. The higher specificity for Con A indicates more interactions between the amino acid residues at the binding site and the carbohydrate ligand than for the pea and lentil lectin-carbohydrate complexes. The carbohydrate-lectin binding results exhibit enthalpy-entropy compensation in that delta Hb (kJ mol-1) = -1.67 +/- 0.06 x 10(4) + (1.30 +/- 0.12)T(K) delta Sb (J mol-1K-1). Differential scanning calorimetry measurements on the thermal denaturation of the lectins and their carbohydrate complexes show that the Con A tetramer dissociates into monomers, while the pea and lentil lectin dimers dissociate into two submonomer fragments. At the denaturation temperature, one carbohydrate binds to each monomer of Con A and the pea and lentil lectins. Complexation with the carbohydrate increases the denaturation temperature of the lectin and the magnitude of the increases yield binding constants in agreement with the determinations from titration calorimetry.

Cultivo da lentilha (Lens culinaris Medik).

Introdução. Clima. Solos e adubação. Cultivares. Plantio. irrigação. Controle de plantas daninhas. Pragas e doenças. Colheita. Custo de produção da lentilha no planalto central (para 1 ha).

ISOLATION AND IN-VITRO HYDROLYSIS OF GLOBULIN G1 FROM LENTILS (LENS-CULINARIS, MEDIK)

The major globulin fraction from lentil seeds was investigated with respect td in vitro hydrolysis by trypsin and chymotrypsin. Globulin was isolated by a NaCl-ascorbate extraction procedure and purified by DEAE-cellulose chromatography and gelfiltration chromatography on Sepharose CL-6B. The purity and identification of the protein were performed by PAGE. The native globulin, with a molecular weight of 375 kD, was resolved by SDS-PAGE into twelve polypeptides with molecular weights ranging from 61 to 14.5 kD. Native and heated globulin GI was hydrolyzed with trypsin and chymotrypsin. SDS-PAGE indicated that native globulin was more resistant to digestion than heated protein. Amino acid analysis of the major globulin revealed that glutamic acid was present in the largest concentration, followed by aspartic acid, arginine and leucine. As is also the case for other legumin-like globulins, lentil GI was deficient in sulfur-containing amino acids.

Estudo fenotípico da região Aorta-Gônada-Mesonefros, de embriões de galinhas Gallus gallus domesticus, com ênfase na aorta dorsal

Acredita-se que os primeiros progenitores da hematopoese definitiva surjam da diferenciação do endotélio da aorta dorsal, na altura da região da Aorta-Gônada-Mesonefros (AGM). Com o intuito de estudar esta região e o fenótipo das células do endotélio da aorta dorsal nesta posição topográfica, ovos galados de Gallus gallus domesticus L. foram incubados em chocadeira, classificados em estádios de E16 a E25 e processados histotecnologicamente para obtenção de secções seriadas na altura da região AGM. Estas passaram por coloração por Hematoxilina-Eosina, histoquímica para PAS, PAS-diastase e Alcian Blue pH 1.0 e pH 2.5, histoquímica por lectinas fluoresceinadas e imunofluorescência para moléculas de superfície, citoesqueleto e matriz extracelular. Foi observada hipertrofia endotelial no assoalho da aorta nos estádios observados, o qual se apresentava positivo ao PAS, com ocorrência frequente de vacuolizações basais PAS negativas, e o surgimento ocasional de grupamentos celulares intravasculares. Nestes, as células que se destacavam da membrana basal do endotélio expressavam progressivamente mais material PAS positivo, o qual, no entanto, em nenhum momento pareceu se tratar de glicogênio. Em relação às glicosaminoglicanas, notamos a presença predominante de ácido hialurônico por todo o mesênquima da região e em outras estruturas como periferia da notocorda, tubo neural e mesoderma lateral. Ocorreu co-expressão de fibronectina e α-actina de músculo liso em células circunjacentes à aorta, na face ventral do vaso. GFAP e BMP-4 são expressas entre as células do tubo neural e em sua periferia, assim como na notocorda do embrião. As lectinas Abrus precatorius, Lens culinarise Ricinus communis mostraram-se positivas principalmente na região subedotelial do assoalho da aorta nos estádios observados neste trabalho. Bandeiraea simplicifolia exibiu pouca marcação na aorta dorsal e a Arachis hypogeae foi negativa. Outras estruturas da região AGM também expressaram resíduos de açúcares revelados por estas lectinas, tais como: notocorda, tubo neural, mesênquima, intestino primitivo e saco vitelínico. Estes resultados acrescentam elementos morfológicos e bioquímicos ao conhecimento sobre a região AGM de embriões de galinha e sobre o endotélio, possivelmente hemogênico, da aorta dorsal.

PGPRs and nitrogen-fixing legumes: a perfect team for efficient Cd phytoremediation?

Cadmium (Cd) is a toxic, biologically non-essential and highly mobile metal that has become an increasingly important environmental hazard to both wildlife and humans. In contrast to conventional remediation technologies, phytoremediation based on legume rhizobia symbiosis has emerged as an inexpensive decontamination alternative which also revitalize contaminated soils due to the role of legumes in nitrogen cycling. In recent years, there is a growing interest in understanding symbiotic legume rhizobia relationship and its interactions with Cd. The aim of the present review is to provide a comprehensive picture of the main effects of Cd in N-2-fixing leguminous plants and the benefits of exploiting this symbiosis together with plant growth promoting rhizobacteria to boost an efficient reclamation of Cd-contaminated soils.

Effect of lentil tannins on albumin hydrolysis by trypsin

Os taninos da casca da semente de lentilha foram extraídos e purificados, levados à interação com albumina isolada de lentilha e com caseína; e estudados por turbidimetria. As interações da albumina e caseína com taninos purificados, a várias relações tanino-proteína, mostraram ser independente e dependente do pH, respectivamente. Hidrólise in vitro com tripsina das proteínas sem taninos indicou que o aquecimento a 99°C/15 min reduzia a susceptibilidade da albumina e aumentava a da caseína à tripsina. A influência de diferentes relações tanino:proteína (1:40; 1:20; 1:5; 1:2,5) na hidrólise mostrou maior inibição para caseína que para albumina de lentilha, independente de aquecimento. Após aquecimento ambas proteínas foram mais hidrolizadas para qualquer das relações tanino proteínas estudadas. A eletroforese em gel de poliacrilamida-dodecilsulfato de sódio do transcurso da hidrólise da interação tanino-albumina nativa mostra a dependência da relação tanino:proteína.

LENTIL TANNIN-GLOBULIN INTERACTIONAND and IN VITRO HYDROLYSIS

As frações protéicas foram isoladas dos cotiledones e os taninos isolados e purificados da casca da lentilha. A fração globulina correspondeu a 42,7 % do nitrogenio total da farinha de lentilha representando a fração protéica majoritária. Comparativamente ao metanol e metanol-HCl 1% a mistura acetona:água (7:3) representou o melhor meio extrator para os taninos da casca. A fração globulina isolada, nativa e aquecida (99oC/15 min), e caseína foram hidrolisadas com tripsina e pepsina na ausência de taninos e na presença de relações tanino:proteína de 1:40, 1:20, 1:10, 1:5 e 1:2,5. A hidrólise tríptica e péptica das proteínas não-aquecidas foram reduzidas com o aumento da relação tanino-proteína. A caseína não aquecida mostrou ser mais susceptível à tripsina que à globulina, o oposto sendo observado com a pepsina. O aquecimento seguido de interação com os taninos e hidrólise teve um efeito mais pronunciado sobre a digestão com tripsina que com pepsina para ambas proteínas.

Role of roscovitine and IBMX on kinetics of nuclear and cytoplasmic maturation of bovine oocytes in vitro

The 3-isobutyl-1-methylxanthine (IBMX) is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte, and roscovitine, a purine known to specifically inhibit MPF kinase activity, maintains bovine oocytes at the germinal vesicle (GV) stage. The present study was conducted to analyze whether cytoplasmic maturation (examined by the pattern of cortical granule (CG) distribution) of bovine oocytes is improved during meiotic arrest with IBMX and roscovitine. Oocytes were matured in vitro in a 10% Knockout(SR) supplemented TCM-199 medium (Control) with either 0.5 mM IBMX or 25 mu M roscovitine (ROSC). Oocytes were stained with fluorescein isothiocyanate conjugated Lens culinaris agglutinin (FITC-LCA) for CG evaluation and with Hoechst 33342 for nuclear stage assessment. At 16 h of culture, the percentage of oocytes remaining in the GV stage was higher (P < 0.05) in the ROSC group (32.41%) compared with the Control and IBMX groups (8.61% and 9.73%, respectively). At 24h of culture, progression of meiosis to M II stage was retarded (P < 0.05) in the ROSC group (24.05%) compared to the Control (60.20%), whereas the IBMX group (33.88%) showed no significant difference to the other two groups. At 16h of maturation, the proportion of oocytes with CG in clusters (immature cytoplasm) was similar between the groups, as was the percentage of peripheral CG (mature) at 24h of maturation. The results of the present study demonstrated that the meiotic inhibitors IBMX and roscovitine delay the progression of nuclear maturation without affecting cytoplasmic maturation, assessed by the analysis of CG repositioning. (c) 2006 Elsevier B.V. All rights reserved.

Efeito do fator de crescimento IGF-I sobre a maturação in vitro de oócitos caninos (Canis familiaris): avaliação da maturação nuclear e citoplasmática

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Metagenomic Anlaysis of microsymbiont selection by the legume plant host

Rhizobium leguminosarum bv.viciae is able to establish nitrogen-fixing symbioses with legumes of the genera Pisum, Lens, Lathyrus and Vicia. Classic studies using trap plants (Laguerre et al., Young et al.) provided evidence that different plant hosts are able to select different rhizobial genotypes among those available in a given soil. However, these studies were necessarily limited by the paucity of relevant biodiversity markers. We have now reappraised this problem with the help of genomic tools. A well-characterized agricultural soil (INRA Bretennieres) was used as source of rhizobia. Plants of Pisum sativum, Lens culinaris, Vicia sativa and V. faba were used as traps. Isolates from 100 nodules were pooled, and DNA from each pool was sequenced (BGI-Hong Kong; Illumina Hiseq 2000, 500 bp PE libraries, 100 bp reads, 12 Mreads). Reads were quality filtered (FastQC, Trimmomatic), mapped against reference R. leguminosarum genomes (Bowtie2, Samtools), and visualized (IGV). An important fraction of the filtered reads were not recruited by reference genomes, suggesting that plant isolates contain genes that are not present in the reference genomes. For this study, we focused on three conserved genomic regions: 16S-23S rDNA, atpD and nodDABC, and a Single Nucleotide Polymorphism (SNP) analysis was carried out with meta / multigenomes from each plant. Although the level of polymorphism varied (lowest in the rRNA region), polymorphic sites could be identified that define the specific soil population vs. reference genomes. More importantly, a plant-specific SNP distribution was observed. This could be confirmed with many other regions extracted from the reference genomes (data not shown). Our results confirm at the genomic level previous observations regarding plant selection of specific genotypes. We expect that further, ongoing comparative studies on differential meta / multigenomic sequences will identify specific gene components of the plant-selected genotypes

The Pangenome of the Plant-Selected Rhizobium leguminosarum bv viciae Sub-popuations

Rhizobium leguminosarum bv viciae (Rlv) is a bacterium able to establish effective symbioses with four different legume genera: Pisum, Lens, Lathyrus and Vicia. Classic studies using trap plants have previously shown that, given a choice, different plants prefer specific genotypes of rhizobia, which are adapted to the host (1, 2). In previous work we have performed a Pool-Seq analysis bases on pooled DNA samples from Rlv nodule isolates obtained from Pisum sativum, Lens culinaris, Vicia fava and V. sativa plants, used as rhizobial traps. This experiment allowed us to test the host preference hypothesis: different plant hosts select specific sub-populations of rhizobia from the available population present in a given soil. We have observed that plant-selected sub-populations are different at the single nucleotide polymorphism (SNP) level. We have selected individual isolates from each sub-population (9 fava-bean isolates, 14 pea isolates 9 vetch isolates and 9 lentil isolates) and sequenced their genomes at draft level (ca. 30x, 90 contigs). Genomic analyses have been carried out using J-species and CMG-Biotools. All the isolates had similar genome size (7.5 Mb) and number of genes (7,300). The resulting Average Nucleotide Identity (ANIm) tree showed that Rhizobium leguminosarum bv viciae is a highly diverse group. Each plant-selected subpopulation showed a closed pangenome and core genomes of similar size (11,500 and 4,800 genes, respectively). The addition of all four sub-population results in a larger, closed pangenome of 19,040 genes and a core genome of similar size (4,392 genes). Each sub-population contains a characteristic set of genes but no universal, plant-specific genes were found. The core genome obtained from all four sub-populations is probably a representative core genome for Rhizobium leguminosarum, given that the reference genome (Rhizobium leguminosarum bv. viciae strain 3841) contains most of the core genome. We have also analyzed the symbiotic cluster (nod), and different nod cluster genotypes were found in each sub-population. Supported by MINECO (Consolider-Ingenio 2010, MICROGEN Project, CSD2009-00006).