Characterization of Exopolysaccharides Produced by Rhizobia Species

ABSTRACT Increasing attention has been given, over the past decades, to the production of exopolysaccharides (EPS) from rhizobia, due to their various biotechnological applications. Overall characterization of biopolymers involves evaluation of their chemical, physical, and biological properties; this evaluation is a key factor in understanding their behavior in different environments, which enables researchers to foresee their potential applications. Our focus was to study the EPS produced by Mesorhizobium huakuii LMG14107, M. loti LMG6125, M. plurifarium LMG11892,Rhizobium giardini bv. giardiniH152T, R. mongolense LMG19141, andSinorhizobium (= Ensifer)kostiense LMG19227 in a RDM medium with glycerol as a carbon source. These biopolymers were isolated and characterized by reversed-phase high-performance liquid chromatography (RP-HPLC), Fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR) spectroscopies. Maximum exopolysaccharide production was 3.10, 2.72, and 2.50 g L-1for the strains LMG6125, LMG19227, and LMG19141, respectively. The purified EPS revealed prominent functional reactive groups, such as hydroxyl and carboxylic, which correspond to a typical heteropolysaccharide. The EPS are composed primarily of galactose and glucose. Minor components found were rhamnose, glucuronic acid, and galacturonic acid. Indeed, from the results of techniques applied in this study, it can be noted that the EPS are species-specific heteropolysaccharide polymers composed of common sugars that are substituted by non-carbohydrate moieties. In addition, analysis of these results indicates that rhizobial EPS can be classified into five groups based on ester type, as determined from the 13C NMR spectra. Knowledge of the EPS composition now facilitates further investigations relating polysaccharide structure and dynamics to rheological properties.

Caracterização das O-acetil-(4-O-metilglicurono)xilanas isoladas da madeira de Eucalyptus urograndis

The O-acetyl-4-O-methyl-(glucurono)xylans were isolated from E. urograndis by extraction with dimethyl sulfoxide, analysed for monosaccharide composition and structurally characterized by NMR spectroscopy. These xylans contained one 4-O-methyl-glucuronic acid substituent and 5.5 acetyl groups for approximately 10 xylose residues. About 10% of 4-O-methyl-glucuronic acid (MeGlcA) units were branched at O-2. The O-acetyl-4-O-methyl-(glucurono)-xylans were composed of the following (1 → 4)-linked β-D-xylopyranosyl structural elements: unsubstituted (51 mol%), 2-O-acetylated (12 mol%), 3-O-acetylated (20 mol%), 2,3-di-O-acetylated (6 mol%) and [MeGlcA α-(1 → 2)] [3-O-acetylated] (11 mol%). The weight-average molar mass and polydispersity of this xylan were 34.9 kDa and 1.16, respectively, as measured by size-exclusion chromatography.

Purification and characterization of a phytoalexin elicitor from spores of the saprobe Mucor ramosissimus

Plants accumulate antimicrobial compounds (phytoalexins) in response to a wide variety of microorganisms. Mucor ramosissimus Samutsevitsch is a saprobe capable of inducing phytoalexin production in soybean cotyledons and in the leaves of tropical Rubiaceae on whose surface it has been found. In the present study, the elicitor from M. ramosissimus was partially purified and the activity compared to that of a glucan elicitor isolated from Phytophthora sojae. Optimal isolation of the elicitor (based on fungal growth, yield of spores and elicitor activity) was achieved by autoclaving spores obtained from nine day-old cultures of the fungus. The elicitor was precipitated with ethanol and purified by chromatography on an anion exchange column, which retained the elicitor, and a Concanavalin A-affinity matrix, to which the elicitor did not bind. The purification resulted in a considerable increase (six-fold) in the specific activity of the elicitor. Neutral sugar composition, analyzed by HPLC, revealed the predominance of mannose, followed by glucose and galactose, whereas colorimetric quantification showed the presence of uronic acids. GC-MS analysis of the elicitor revealed the predominance of glucuronic acid and mannose. These results suggest that fragments of mucoran-type polysaccharides are the phytoalexin elicitors present in the spores of the saprobe M. ramosissimus. Our results also indicate for the first time that soybean cotyledon tissues can recognize fragments of glucuronic-acid heteropolymers as phytoalexin elicitors.

Galactosaminoglycans from normal myometrium and leiomyoma

In many tumors, the amount of chondroitin sulfate in the extracellular matrix has been shown to be elevated when compared to the corresponding normal tissue. Nevertheless, the degree of chondroitin sulfate increase varies widely. In order to investigate a possible correlation between the amount of chondroitin sulfate and tumor size, several individual specimens of human leiomyoma, a benign uterine tumor, were analyzed. The glycosaminoglycans from eight tumors were extracted and compared with those from the respective adjacent normal myometrium. The main glycosaminoglycan found in normal myometrium was dermatan sulfate, with small amounts of chondroitin sulfate and heparan sulfate. In leiomyoma, both dermatan sulfate and chondroitin sulfate were detected and the total amounts of the two galactosaminoglycans was increased in all tumors when compared to normal tissue. In contrast, the heparan sulfate concentration decreased in the tumor. To assess the disaccharide composition of galactosaminoglycans, these compounds were incubated with bacterial chondroitinases AC and ABC. The amounts of L-iduronic acid-containing disaccharides remained constant, whereas the concentration of D-glucuronic acid-containing disaccharides increased from 2 to 10 times in the tumor, indicating that D-glucuronic acid-containing disaccharides are responsible for the elevation in galactosaminoglycan concentration. This increase is positively correlated with tumor size.

Distribution of small proteoglycans and glycosaminoglycans in humerus-related articular cartilage of chickens

The expression of components present in the cartilaginous extracellular matrix is related to development, gender, and genotype, as well as to the biomechanical properties of each type of cartilage. In the present study, we analyzed small proteoglycans and glycosaminoglycans present in different cartilages of the chicken wing after extraction with guanidine hydrochloride or papain. Quantitative analysis of glycosaminoglycans showed a larger amount in humeral cartilage (around 200 mg/g tissue) than in articular cartilage of the radius and ulna, with 138 and 80 mg/g tissue, respectively. Non-collagenous proteins isolated were predominantly from cartilage in the proximal regions of the humerus and radius. D4 fractions obtained by ultracentrifugation were separated by DEAE-Sephacel and Octyl-Sepharose chromatography and analyzed by SDS-PAGE. Two bands of 57 and 70-90 kDa were observed for all samples treated with ß-mercaptoethanol. Immunoblotting of these proteins was positive for the small proteoglycans fibromodulin and decorin, respectively. Apparently, the 57-kDa protein is present in macromolecular complexes of 160 and 200 kDa. Chondroitin sulfate was detected in all regions. HPLC analysis of the products formed by chondroitinase AC and ABC digestion mainly revealed ß-D-glucuronic acid and N-acetyl ß-D-galactosamine residues. The 4-sulfation/6-sulfation ratio was close to 3, except for the proximal cartilage of the radius (2.5). These results suggest functional differences between the scapula-humerus, humerus-ulna, and humerus-radius joints of the chicken wing. This study contributes to the understanding of the physiology of cartilage and joints of birds under different types of mechanical stress.

Partial characterization and anticoagulant activity of a heterofucan from the brown seaweed Padina gymnospora

The brown algae Padina gymnospora contain different fucans. Powdered algae were submitted to proteolysis with the proteolytic enzyme maxataze. The first extract of the algae was constituted of polysaccharides contaminated with lipids, phenols, etc. Fractionation of the fucans with increasing concentrations of acetone produced fractions with different proportions of fucose, xylose, uronic acid, galactose, and sulfate. One of the fractions, precipitated with 50% acetone (v/v), contained an 18-kDa heterofucan (PF1), which was further purified by gel-permeation chromatography on Sephadex G-75 using 0.2 M acetic acid as eluent and characterized by agarose gel electrophoresis in 0.05 M 1,3 diaminopropane/acetate buffer at pH 9.0, methylation and nuclear magnetic resonance spectroscopy. Structural analysis indicates that this fucan has a central core consisting mainly of 3-ß-D-glucuronic acid 1-> or 4-ß-D-glucuronic acid 1 ->, substituted at C-2 with alpha-L-fucose or ß-D-xylose. Sulfate groups were only detected at C-3 of 4-alpha-L-fucose 1-> units. The anticoagulant activity of the PF1 (only 2.5-fold lesser than low molecular weight heparin) estimated by activated partial thromboplastin time was completely abolished upon desulfation by solvolysis in dimethyl sulfoxide, indicating that 3-O-sulfation at C-3 of 4-alpha-L-fucose 1-> units is responsible for the anticoagulant activity of the polymer.

Adventitious rooting in cuttings of croton and hibiscus in response to indolbutyric acid and humic acid

Adventitious rooting of ornamental plants can be accelerated by the application of growth regulators, such as auxin. Humic acids, organic matter in soil and organic compounds also have a biostimulant effect. This work evaluated the rooting in cuttings of croton (Codianeum variegatum L. Rumph) and hibiscus (Hibiscus rosa-sinensis L) in response to the application of different concentrations of indolbutyric acid (IBA) and humic acid (HA). The experiment was carried out in a greenhouse. Apical stem cuttings were treated with solutions at concentrations of: 0, 250, 500, 1000, 2000 mg L-1 IBA and 0, 10, 20, 30, 40 mmol L-1 HA carbon isolated from vermicomposting. Forty-five days after the applications, the cuttings were removed from the pots containing carbonized rice hull and the following variables were measured: rooting number, length and width of leaves, fresh and dry matter of root and aerial part and root area. The results were subjected to analysis of variance and the qualitative and quantitative effects of the treatments were compared by contrast and regression, respectively. Regression equations were used to determine the maximum efficiency level of root dry matter according to IBA and HA. Higher accumulation of root dry matter was recorded for the treatments with the doses 579 mg L-1 IBA and 14 mmol L-1 HA and 970 mg L-1 IBA and 50 mmol L-1 HA for root cuttings of croton and hibiscus, respectively. It was found that the application of eiher IBA or HA at the indicated doses accelerates rooting in cuttings of croton and hibiscus and contributes to the formation of vigorous plants.

Effect of acetic acid on rice seeds coated with rice husk ash

Flooded rice cultivation promotes anaerobic conditions, favoring the formation of short chain organic acids such as acetic acid, which may be toxic to the crop. The objective of this study was to evaluate the effect of acetic acid on rice seeds coated with rice husk ash. The experiment was arranged in a 2 x 5 x 5 factorial randomized design, with two cultivars (IRGA 424 and BRS Querência), five doses of coating material (0, 2, 3,4 e 5 g kg-1 seed) and five concentrations of acetic acid (0, 3, 6, 9 and 12 mM), with 4 replications, totaling 50 treatments. The variables first count of germination, germination, shoot and root length, dry weight of shoots and roots were recorded. The results showed that coating rice seeds with rice husk ash up to 5 g kg-1 seed does not influence the performance of rice seeds of cultivars IRGA 424 and BRS Querência when exposed to concentrations of 12 mM acetic acid. The presence of acetic acid in the substrates used for seed germination reduced the vigor and viability of seeds of cultivars IRGA 424 and BRS Querência, as well as seedling development, affecting mainly the roots of BRS Querência.

Heat shock and salicylic acid on postharvest preservation of organic strawberries

Heat shock and salicylic acid have been studied on shelf-life extension of fruits. The benefits of these techniques have been related to their effect on inducing physiological defense responses against the oxidative stress and pathogen development. The objective of this study was to evaluate the effect of heat shock and salicylic acid on the postharvest preservation and contents of total phenolics, anthocyanins, ascorbic acid, fresh weight loss and microbiological quality of organic strawberries cv. Dover. Strawberries produced organically and stored at 5 ºC were subjected to heat shock (45 ºC ± 3 ºC for 3 h), application of salicylic acid (soaking in 2.0 mmol L-1 solution), heat shock in combination with salicylic acid and control. After treatment, the fruits were packed and stored in a climatic chamber at 5 ºC ± 2 ºC. At 1, 7 and 14 days, the experimental units were removed from refrigeration and kept at room temperature of approximately 20 ºC for two days. There was no effect of treatments on fresh weight loss, incidence of pathogens or chemical variations in strawberry fruits during the storage period. In natural conditions, organically grown strawberries remained in good condition for sale up to seven days of storage in all treatments.

Application of GA3 and girdling of branches on the production of extemporaneous fruits of 'Tahiti' acid lime

The objective of this study was to evaluate the effect of girdling of branches and the application of GA3, still in the main crop flowering on the formation of reproductive structures, the fruit set of the following extemporaneous flowering of 'Tahiti' acid lime. This work used a randomized block design with five replications in a 4x3 factorial design. The first factor was the foliar application of GA3, 88 days before extemporaneous flowering at five concentrations (0, 7, 14 and 21 mg.L-1). The second factor was the girdling times, 108 days before flowering onset (A108AF), 78 days before flowering (A78AF) plus no-girdling control (NG). In extemporaneous flowering, the proportion of buds, flowers and fruits that abscised was 38.75, 31.25 and 18.64% respectively and 11.34% of all reproductive structures formed were collected. GA3 did not affect fruit set. Girdling increased fruit set by 229 and 256% at A108DAF and A78DAF respectively, in relation to NG plants. The GA3 reduced the formation of floral structures, which resulted in a quadratic increase in field production by reducing the competition for photoassimilates among fruits. The concentration of 21 mg.L-1 was the most productive, with 15.97 fruit.plant-1.

Acclimation of croton and hibiscus seedlings in response to the application of indobultiric acid and humic acid for rooting

The vegetative propagation of ornamental plants can be accelerated by applying plant growth regulators. Amongst them, the use of auxins, plant hormones with physiological effects on cell elongation and rooting have stood out. Alternatively, the application of humic acids, bioactive fraction of soil organic matter, also results in increases in rooting cuttings of ornamental plants. The objective of this work was to study the growth characteristics and the nutritional contents of croton and hibiscus plants during acclimation of seedlings in response to different concentrations of indolebutyric acid (IBA) and humic acid (HA) applied to cuttings for rooting. The experiment was conducted in greenhouse, and the apical stem cuttings were treated with solutions with concentrations of 0, 250, 500, 1000 and 2000 mg L-1of IBA and 0, 10, 20, 30 and 40 mg L-1 of C from HA. At 45 days of rooting in carbonized rice husk, they were individually transferred to plastic bags of 2.0 dm3 containing a mixture of soil: sand: manure (2: 1: 1) as substrate. At 90 days of acclimation, the plants were collected for measurement of growth and nutritional variables. The results showed that the application of the IBA stimulates the absorption of nutrients and growth of croton cuttings and transplanted hibiscus, contributing to formation of vigorous seedlings. A similar response occurred with the application of HA in hibiscus cuttings

Hippuric acid in urine: reference values

OBJECTIVE: To establish reference values for hippuric acid (HA) excreted in the urine, and to evaluate the impact of age, gender, alcohol, and tobacco, on these levels in a population nonexposed to toluene. METHODS: Reference values for hippuric acid in urine were determined in 115 toluene nonexposed healthy volunteers, from Alfenas city, Southeastern Brazil. A questionnaire was applied to each volunteer and data on occupational and personal habits were collected. Biochemical and hematological analyses were used to confirm the volunteers' good health condition. Reference values were expressed in g HA/g urine creatinine, as mean ± standard deviation (x ± SD), median, 95% confidence interval (95%CI), 95th percentile, and upper reference value (URV, mean +2 SD). RESULTS: Reference values of hippuric acid in urine were: mean ± standard deviation =0.18±0.10; median =0.15; 95% confidence interval =0.16±0.20; 95th percentile = 0.36 and upper reference value (URV, mean +2 SD) =0.38. Statistically significant differences in urinary HA (Wilcoxon - Mann/Whitney, p<0.05) were observed for different genders and age groups. Alcohol ingestion and smoking habit did not significantly affect the results. CONCLUSIONS: The reference values of hippuric acid in urine can be used in biomonitoring programs of workers occupationally exposed to toluene, especially in the southern region of the state of Minas Gerais. Age and gender may affect the HA reference values.

Evidence of inadequate docosahexaenoic acid status in Brazilian pregnant and lactating women

Recently published data concerning dietary intake of fat and food sources of (n-3) long-chain polyunsaturated fatty acids (LCPUFA) in Brazil are reviewed together with data on biochemical indices of PUFA status during pregnancy and lactation and PUFA composition of breast milk in Brazilian adolescents and adults. Potential inadequacies of docosahexaenoic acid (DHA) status among Brazilian pregnant and lactating women have not yet been thoroughly evaluated. The data reviewed show that dietary intake of food sources of n-3 LCPUFA is low and possibly deficient in Brazil, and that biochemical indices of maternal DHA status and breast milk DHA content are low compared to the international literature. These data indicate inadequate DHA status among Brazilian women during pregnancy and lactation, but this evidence needs confirmation through comprehensive and specific population-based studies.

Trans fatty acid intake among the population of the city of São Paulo, Brazil

OBJECTIVE: To analyze the monounsaturated and polyunsaturated trans fatty acid intake among the general population. METHODS: A cross-sectional study was conducted in São Paulo, Southeastern Brazil, in 2003, on a representative sample of 2,298 male and female subjects, including 803 adolescents (12 to 19 years), 713 adults (20 to 59 years) and 782 elderly people (60 years or over). Food intake was measured using 24-hour recall. Mean trans fatty acid intake was described according to gender and age group. RESULTS: The mean trans fatty acid intake was 5.0 g/day (SE = 0.1), accounting for 2.4% (SE = 0.1) of total energy and 6.8% (SE = 0.1) of total lipids. The adolescents had the highest mean intake levels (7.4 g/day; 2.9% of energy) while the adults and the elderly had similar intake (2.2% of energy for both; 6.4% of lipids and 6.5% of lipids, respectively). The mean trans fatty acid intake among adult and elderly women (approximately 2.5% of energy and 7.0% of lipids) was higher than among men in the same age group. The food item with the highest contribution towards trans fatty acids was margarine, accounting for more than 30% of total intake, followed by filled cookies among adolescents and meat among adults and the elderly. CONCLUSIONS: The trans fatty acid intake is above the level recommended by the World Health Organization. Replacement of the trans fatty acids in manufactured food items may be an effective measure for reducing trans fatty acid intake in Brazil.

Gastric acid secretion response in the Cebus apella: a monkey model of chronic Chagas disease

The objective was to study the secretory pattern, both basal and stimulated either by histamine (0.1 mg/kg) or pentagastrin (64 ug/kg) in eighteen Cebus apella monkeys chronically infected with different T. cruzi strains (CA1, n=10; Colombian, n=4 and Tulahuen, n=4) and to describe the morphological findings in the gastrointestinal tract in twelve infected (6 sacrificed and 6 spontaneously dead) and four healthy monkeys. All infected monkeys and 35 healthy ones were evaluated by contrast X-ray examination. No differences were observed in basal acid output between control and infected groups. Animals infected with the Tulahuen and Colombian strains showed significant lower values of peak acid output in response to histamine or pentagastrin (p<0.01 and p<0.05 respectively; "t" test) in comparison to the controls. Barium contrast studies showed enlargement and dilatation of the colon in three infected animals. Histopathological lesions were seen in 75% of the autopsied animals either in colon alone (33%) or both, in colon and esophagus (42%). The normal secretion observed in the CA1 infected group could be due to a lower virulence of the strain, a lower esophagic tropism or the necessity of a longer post-infection time to cause lesions.