Metabolomic exhibits different profiles and potential biomarkers of Vitis spp. co-cultivated with Fusarium oxysporum for short, medium, and long times.

Differential rootstock tolerance to Fusarium spp. supports viticulture worldwide. However, how plants stand against the fungus still needs to be explored. We hypothesize it involves a differential metabolite modulation. Thus, we performed a gas chromatography coupled with mass spectrometry (GC-MS) analysis of Paulsen P1103 and BDMG573 rootstocks, co-cultured with Fusarium oxysporum (FUS) for short, medium, and long time (0, 4, and 8 days after treatment [DAT]). In shoots, principal component analysis (PCA) showed a complete overlap between BDMG573 non-co-cultivated and FUS at 0 DAT, and P1103 treatments showed a slight overlap at both 4 and 8 DAT. In roots, PCA exhibited overlapping between BDMG573 treatments at 0 DAT, while P1103 treatments showed overlapping at 0 and 4 DAT. Further, there is a complete overlapping between BDMG573 and P1103 FUS profiles at 8 DAT. In shoots, 1,3-dihydroxyacetone at 0 and 4 DAT and maltose at 4 and 8 DAT were biomarkers for BDMG573. For P1103, glyceric acid, proline, and sorbitol stood out at 0, 4, and 8 DAT, respectively. In BDMG573 roots, the biomarkers were ß-alanine at 0 DAT, cellobiose and sorbitol at both 4 and 8 DAT. While in P1103 roots, they were galactose at 0 and 4 DAT and 1,3-dihydroxyacetone at 8 DAT. Overall, there is an increase in amino acids, glycolysis, and tricarboxylic acid components in tolerant Paulsen P1103 shoots. Thus, it provides a new perspective on the primary metabolism of grapevine rootstocks to F. oxysporum that may contribute to strategies for genotype tolerance and early disease identification.

Assessment of different systems for the production of aldonic acids and sorbitol by calcium alginate-immobilized Zymomonas mobilis cells.

Equimolar amounts of lactobionic acid and sorbitol may be obtained in a reaction catalyzed by the enzymes glucose-fructose oxidoreductase and glucono-δ-lactonase, which are found in the periplasm of Zymomonas mobilis. These reactions are generally conducted using immobilized bacterial cells, and the cell treatment and immobilization steps are costly and time-consuming. This study evaluated alternatives to simplify the preparation of calcium alginate-immobilized biocatalyst and its application in different operation modes and types of reactors. It was possible to eliminate cell permeabilization with cetyltrimethylammonium bromide, and the reticulation of Z. mobilis cells with glutaraldehyde sufficed to inhibit the fermentative metabolism of carbohydrates by the bacterium, with accumulation of bioconversion products. When the process was carried out in a mechanically stirred reactor in batch mode, 530 mmol L- 1 of products were obtained in 24 h. The process was also tested in fed-batch mode so as to use of a larger amount of lactose, since it could not be used in the batch because of its low solubility in water. Under this condition, final products concentration reached 745 mmol L- 1 within 42 h. Similar results were obtained for reactions conducted in a pneumatically stirred reactor in batch and fed-batch modes, proving the potential use of this process in several industrial settings.

Effects of different osmolarities on bacterial biofilm formation.

Biofilm formation depends on several factors. The influence of different osmolarities on bacterial biofilm formation was studied. Two strains (Enterobacter sp. and Stenotrophomonas sp.) exhibited the most remarkable alterations. Biofilm formation is an important trait and its use has been associated to the protection of organisms against environmental stresses.

Effects of different osmolarities on bacterial biofilm formation

Biofilm formation depends on several factors. The influence of different osmolarities on bacterial biofilm formation was studied. Two strains (Enterobacter sp. and Stenotrophomonas sp.) exhibited the most remarkable alterations. Biofilm formation is an important trait and its use has been associated to the protection of organisms against environmental stresses.

Effects of different osmolarities on bacterial biofilm formation

Biofilm formation depends on several factors. The influence of different osmolarities on bacterial biofilm formation was studied. Two strains (Enterobacter sp. and Stenotrophomonas sp.) exhibited the most remarkable alterations. Biofilm formation is an important trait and its use has been associated to the protection of organisms against environmental stresses.

Cloning, expression and optimized production in a bioreactor of bovine chymosin B in Pichia (Komagataella) pastoris under AOX1 promoter.

The codon sequence optimized bovine prochymosin B gene was cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and integrated into the genome of the methylotrophic yeast Pichia (Komagataella) pastoris (P. pastoris) strain GS115. A transformant clone that showed resistance to over 4 mg G418/ml and displayed the highest milk-clotting activity was selected. Cell growth and recombinant bovine chymosin production were optimized in flask cultures during methanol induction phase achieving the highest coagulant activity with low pH values, a temperature of 25°C and with the addition of sorbitol and ascorbic acid at the beginning of this period. The scaling up of the fermentation process to lab-scale stirred bioreactor using optimized conditions, allowed to reach 240 g DCW/L of biomass level and 96 IMCU/ml of milk-clotting activity. The enzyme activity corresponded to 53 mg/L of recombinant bovine chymosin production after 120 h of methanol induction. Western blot analysis of the culture supernatant showed that recombinant chymosin did not suffer degradation during the protein production phase. By a procedure that included high performance gel filtration chromatography and 3 kDa fast ultrafiltration, the recombinant bovine chymosin was purified and concentrated from fermentation cultures, generating a specific activity of 800 IMCU/Total Abs(280 nm) and a total activity recovery of 56%. This study indicated that P. pastoris is a suitable expression system for bioreactor based fed-batch fermentation process for the efficient production of recombinant bovine chymosin under methanol-inducible AOX1 promoter.

Shoot-tip cryopreservation by droplet vitrification of Byrsonima intermedia A. Juss.: a woody tropical and medicinal plant species from Brazilian cerrado.

Cryopreservation of plant species is poorly investigated in Brazil. The aim of this study was to cryopreserve Byrsonima intermedia shoot apical meristems through droplet vitrification. A culture medium for shoot-tips growth was established using the Woody Plant Medium supplemented with 2.22 uM 6-benzylaminopurine. Excised shoot-tips were subjected to pre-culture and/or post-culture treatments on Murashige and Skoog medium with 0.3 M sucrose for 24 h prior dehydration on PVS2 at 0°C for 15, 30 or 45 minutes prior to plunging in liquid nitrogen. The effect of 15 days of shoot pre-growth on a high osmotic medium (0.3 M sucrose or 0.21 M sorbitol + 0.09 M sucrose) prior to meristem excision and cryopreservation was also investigated. Pre-culturing shoot-tips on 0.3 M sucrose for 24 h prior to cryopreservation increased the regrowth level after thawing to 90%. Shoot-tips excised from shoots pre-grown on MS + 0.21 M sorbitol + 0.09 M sucrose for 15 days presented a satisfactory regrowth level (67%).

Association study of sorbitol dehydrogenase -888G>C polymorphism with type 2 diabetic retinopathy in Caucasian-Brazilians.

Diabetic retinopathy (DR) is a common chronic complication of diabetes and remains the leading cause of blindness in working-aged people. Hyperglycemia increases glucose flux through the polyol pathway, in which aldose reductase converts glucose into intracellular sorbitol, which is subsequently converted to fructose by sorbitol dehydrogenase (SDH). The accelerated polyol pathway triggers a cascade of events leading to retinal vascular endothelial dysfunction and the eventual development of DR. Polymorphisms in the gene encoding aldose reductase have been consistently associated with DR. However, only two studies have analyzed the relationship between polymorphisms in the gene encoding SDH (SORD) and DR. In this case-control study, we investigated whether the -888G > C polymorphism (rs3759890) in the SORD gene is associated with the presence or severity of DR in 446 Caucasian-Brazilians with type 2 diabetes (241 subjects with and 205 subjects without DR). The -888G > C polymorphism was also examined in 105 healthy Caucasian blood donors, and the genotyping of this polymorphism was carried out by real-time PCR. The genotype and allele frequencies of the -888G > C polymorphism in patients with type 2 diabetes were similar to those of blood donors (G allele frequency = 0.16 in both groups of subjects). Similarly, the genotype and allele frequencies in patients with DR or the proliferative form of DR were similar to those of patients without this complication (P > 0.05 for all comparisons). Thus, our findings suggest that the -888G > C polymorphism in the SORD gene is not involved in the pathogenesis of DR in type 2 diabetes.

Sorbitol dehydrogenase is a cytosolic protein required for sorbitol metabolism in Arabidopsis thaliana.

Sorbitol is converted to fructose in Rosaceae species by SORBITOL DEHYDROGENASE (SDH, EC 1.1.1.14), especially in sink organs. SDH has also been found in non-Rosaceae species and here we show that the protein encoded by At5g51970 in Arabidopsis thaliana (L.) Heynh. possesses the molecular characteristics of an SDH. Using a green fluorescent protein-tagged version and anti-SDH antisera, we determined that SDH is cytosolically localized, consistent with bioinformatic predictions. We also show that SDH is widely expressed, and that SDH protein accumulates in both source and sink organs. In the presence of NAD+, recombinant SDH exhibited greatest oxidative activity with sorbitol, ribitol and xylitol as substrates; other sugar alcohols were oxidized to a lesser extent. Under standard growth conditions, three independent sdh- mutants developed as wild-type. Nevertheless, all three exhibited reduced dry weight and primary root length compared to wild-type when grown in the presence of sorbitol. Additionally, under short-day conditions, the mutants were more resistant to dehydration stress, as shown by a reduced loss of leaf water content when watering was withheld, and a greater survival rate on re-watering. This evidence suggests that limitations in the metabolism of sugar alcohols alter the growth of Arabidopsis and its response to drought.

Anti-Candida albicans effectiveness of citral and investigation of mode of action.

CONTEXT: Candidiasis is a mycosis caused by Candida species, which is of clinical importance due to the increase in resistant yeasts. Candida infection has been a serious health problem due to the inappropriate use of antibiotics. Therefore, it is necessary to study molecules with an antifungal action. Citral is a monoterpene with known pharmacological properties, including antimicrobial action. OBJECTIVE: The aim of this work was to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of citral and the probable mode of action. MATERIALS AND METHODS: The MIC of citral was determined by the broth microdilution method using Sabouraud dextrose medium. Additionally, the interference of citral in cell wall (sorbitol assay) and the binding of citral to ergosterol and cholesterol were studied, carried out by broth microdilution method. RESULTS: The MIC and MFC of citral were 512 and 1024 µg/mL, respectively. The MIC of amphotericin B was 1 µg/mL. The mechanism of action did not involve either the cell wall or ergosterol. However, the presence of cholesterol increased the MIC of citral to 1024 µg/mL, indicating there is some interaction between citral and cholesterol. Amphotericin B was used as the positive control, and it showed a high MIC in the presence of ergosterol (32 µg/mL), while in the presence of cholesterol MIC increased to 4 µg/mL. CONCLUSION: Citral inhibits the growth of C. albicans. The probable mechanism of action did not involve the cell wall or ergosterol. Citral is able to interact with cholesterol. More studies are necessary to describe their effects completely.

Stimulating effect of sorbitol and xylitol on germination and growth of some xerophilic fungi.

Sorbitol and xylitol are polyols often used in foods as naturally occurring sugar substitutes. They provide sweet taste and reduced calories in products of intermediate moisture. This type of food is susceptible to spoilage by xerophilic molds which affect shelf life of foods and produce significant losses. The aim of the present work was to study the effect of glycerol, sorbitol and xylitol on the germination and growth of four xerophilic fungi at different temperatures and water activity levels. Penicillium chrysogenum, Wallemia sebi, Eurotium chevalieri and Eurotium repens were cultivated on malt extract agar with the addition of the respective polyols and a(w) adjusted to 0.85, 0.88, 0.90 and 0.93. Incubation was made at 25, 30 and 35 °C. Results of the present study demonstrated that sorbitol and xylitol affect the growth kinetics of the four fungal species. The observed tendency was that these solutes shortened the germination times and increased the growth rates. The effect of each solute depended on the fungal species and the a(w)/temperature combinations. At lower a(w) the influence was more evident on the germination times while the effect on growth rates was more pronounced at higher a(w) levels.

Analysis of experimental errors in bioprocesses. 1. Production of lactobionic acid and sorbitol using the GFOR (glucose-fructose oxidoreductase) enzyme from permeabilized cells of Zymomonas mobilis.

The proper determination of experimental errors in bioprocesses can be very important because experimental errors can exert a major impact on the analysis of experimental results. Despite this, the effect of experimental errors on the analysis of bioprocess data has been largely overlooked in the literature. For this reason, we performed detailed statistical analyses of experimental errors obtained during the production of lactobionic acid and sorbitol in a system utilizing as catalyst the GFOR (glucose-fructose oxidoreductase) enzyme from permeabilized cells of the bacteria Zymomonas mobilis. The magnitude of the experimental errors thus obtained were then correlated with the process operation conditions and with the composition of the culture media used for bacterial growth. It is shown that experimental errors can depend very significantly on the operation conditions and affect the interpretation of available experimental data. More specifically, in this study, experimental errors depended on the nutritional supplements added to the cultivation medium, the inoculation process, and the reaction time, which may be of fundamental importance for actual process development. The results obtained also indicate, for the first time, that GFOR activity can be affected by the composition of the medium in which cells are cultivated.

Skeletal muscle sorbitol levels in diabetic rats with and without insulin therapy and endurance exercise training.

Sorbitol accumulation is postulated to play a role in skeletal muscle dysfunction associated with diabetes. The purpose of this study was to determine the effects of insulin and of endurance exercise on skeletal muscle sorbitol levels in streptozotocin-induced diabetic rats. Rats were assigned to one experimental group (control sedentary, control exercise, diabetic sedentary, diabetic exercise, diabetic sedentary no-insulin). Diabetic rats received daily subcutaneous insulin. The exercise-trained rats ran on a treadmill (1 hour, 5X/wk, for 12 weeks). Skeletal muscle sorbitol levels were the highest in the diabetic sedentary no-insulin group. Diabetic sedentary rats receiving insulin had similar sorbitol levels to control sedentary rats. Endurance exercise did not significantly affect sorbitol levels. These results indicate that insulin treatment lowers sorbitol in skeletal muscle; therefore sorbitol accumulation is probably not related to muscle dysfunction in insulin-treated diabetic individuals. Endurance exercise did not influence intramuscular sorbitol values as strongly as insulin.

Structural and kinetic characterization of a maize aldose reductase.

The aldo-keto reductases (AKRs) are classified as oxidoreductases and are found in organisms from prokaryotes to eukaryotes. The AKR superfamily consists of more than 120 proteins that are distributed throughout 14 families. Very few plant AKRs have been characterized and their biological functions remain largely unknown. Previous work suggests that AKRs may participate in stress tolerance by detoxifying reactive aldehyde species. In maize endosperm, the presence of an aldose reductase (AR; EC 1.1.1.21) enzyme has also been hypothesized based on the extensive metabolism of sorbitol. This manuscript identifies and characterizes an AKR from maize (Zea mays L.) with features of an AR. The cDNA clone, classified as AKR4C7, was expressed as a recombinant His-tag fusion protein in Escherichia coli. The product was purified by immobilized metal affinity chromatography followed by anion exchange chromatography. Circular dichroism spectrometry and SAXS analysis indicated that the AKR4C7 protein was stable, remained folded throughout the purification process, and formed monomers of a globular shape, with a molecular envelope similar to human AR. Maize AKR4C7 could utilize dl-glyceraldehyde and some pentoses as substrates. Although the maize AKR4C7 was able to convert sorbitol to glucose, the low affinity for this substrate indicated that AKR4C7 was probably a minimal contributor to sorbitol metabolism in maize seeds. Polyclonal antisera raised against AKR4C7 recognized at least three AR-like polypeptides in maize kernels, consistent with the presence of a small gene family. Diverse functions may have evolved for maize AKRs in association with specific physiological requirements of kernel development.

Genetic relatedness of a non-motile variant O157 enteropathogenic Escherichia coli (EPEC) strain and E. coli strains belonging to pathogenic related groups.

The study was undertaken to determine the clonal relationship and the genetic diversity among Escherichia coli isolates by comparing a non-motile O157 variant with three O157:H7 EHEC isolates and one O55:H7 enteropathogenic E. coli (EPEC) strain. E. coli strains were characterized by sorbitol phenotype, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, random amplification polymorphic DNA, and the presence of specific virulence genes (stx, E-hly and LEE genes). Sorbitol fermentation was observed in O157:H- (strain 116I), O55:H7 and O157:H7 (strain GC148) serotypes. stx1 or stx2 and E-hly genes were only detected among O157:H7 isolates. LEE typing revealed specific allele distribution: eaegamma, tirgamma, espAgamma, espBgamma associated with EPEC O55:H7 and EHEC O157:H7 strains (B1/1 and EDL 933), eaealpha, tiralpha, espAalpha, espBalpha related to the 116I O157:H- strain and the GC148 strain presented non-typable LEE sequences. Multilocus enzyme profiles revealed two main clusters associated with specific LEE pathotypes. E. coli strains were discriminated by random amplification of polymorphic DNA-polymerase chain reaction and pulsed-field gel electrophoresis methodologies. The molecular approaches used in this study allowed the determination of the genetic relatedness among E. coli strains as well as the detection of lineage specific group markers.

Replacing the general energy-coupling proteins of the phospho-enol-pyruvate: sugar phosphotransferase system of Salmonella typhimurium with fructose-inducible counterparts results in the inability to utilize nonphosphotransferase system sugars.

A Salmonella typhimurium mutant lacking Enzyme I and HPr, general proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), but producing homologues EI(Fructose) and FPr constitutively, did not grow in minimal medium supplemented with non-PTS sugars (melibiose, glycerol, and maltose) in the absence of any trace of Luria-Bertani broth; adding cyclic AMP allowed growth. On melibiose, rapid growth began only when melibiose permease activity had reached a threshold level. Wild-type cultures reached this level within about 2 h, but the mutant only after a 12-14 h lag period, and then only when cyclic AMP had been added to the medium. On a mixture of melibiose and a PTS sugar, permease was undetectable in either the wild type or mutant until the PTS sugar had been exhausted. Permease then appeared, increasing with time, but in the mutant it never reached the threshold allowing rapid growth on melibiose unless cyclic AMP had been added. On rich medium supplemented with melibiose or glycerol, the mutant produced lower (30%) levels of melibiose permease or glycerol kinase compared with the wild type. We propose that poor phosphorylation of the regulatory protein Enzyme IIA(Glucose), leading to constitutive inducer exclusion and catabolite repression in this strain, accounts for these results.

[Study of phenotypical and antimicrobial susceptibility markers in enteric Escherichia coli strains]. / Estudio de marcadores fenotípicos y de susceptibilidad antimicrobiana en cepas de Escherichia coli entéricas.

Forty strains of Escherichia coli isolated from children under 5 years of age with acute diarreas, coming from different provinces of the country , were analyzed. Four important phenotypical determinants were tested: sorbosa, sorbitol, enterohemolysin and 0157:H7 serology, in order to select those strains from enterohemorrhagic or Shiga toxin-producing category. Likewise, they were characterized by biotyping and antimicrobial susceptibility methods. The use of phenotypical tests showed six strains with presumptive characteristics, four of which were most likely to be Shiga toxin-producing strains. In antimicrobial susceptibility test, the strains showed high resistance mainly to ampicillin and trimethrophin-sulfamethoxasole. Another interesting finding were intermediate resistance and susceptibility values to augmentin, aztreonan and ceftriaxone. There were 12 antimicrobial resistance patterns of which 10 were multi-resistant.

Zymomonas mobilis as catalyst for the biotechnological production of sorbitol and gluconic acid.

The conversion of glucose and fructose into gluconic acid (GA) and sorbitol (SOR) was conducted in a batch reactor with free (CTAB-treated or not) or immobilized cells of Zymomonas mobilis. High yields (more than 90%) of gluconic acid and sorbitol were attained at initial substrate concentration of 600 g/L (glucose plus fructose at 1:1 ratio), using cells with glucose-fructose-oxidoreductase activity of 75 U/L. The concentration of the products varied hyperbolically with time according to the equations (GA) = t (GA)max /(W(GA) + t), (SOR) = t (SOR)max/(W(SOR) + t), V(GA) = [W(GA) (GA)max]/(W(GA) + t)2 and V(SOR) = [W(SOR) (SOR)max]/(W(SOR) + t)2. Taking the test carried out with free CTAB-treated cells as an example, the constant parameters were (GA)max = 541 g/L, (SOR)max = 552 g/L, WGA = 4.8 h, W(SOR) = 4.9 h, v(GA) = 112.7 g/L x h and v(SOR) = 112.7 g/L x h.

Zymomonas mobilis as catalyst for the biotechnological production of sorbitol and gluconic acid.

The conversion of glucose and fructose into gluconic acid (GA) and sorbitol (SOR) was conducted in a batch reactor with free (CTAB-treated or not) or immobilized cells of Zymomonas mobilis. High yields (more than 90%) of gluconic acid and sorbitol were attained at initial substrate concentration of 600 g/L (glucose plus fructose at 1:1 ratio), using cells with glucose-fructose-oxidoreductase activity of 75 U/L. The concentration of the products varied hyperbolically with time according to the equations (GA)=t(GA)(max)/(W(GA) +t), (SOR)=t (SOR)(max)/(W(Sor)+t), v(GA)=[W(GA) (GA)(max)]/(W(GA)+t)(2) and V(SOR)=[W(SOR) (SOR)(max)]/(W(SOR)+t)(2). Taking the test carried out with free CTAB-treated cells as an example, the constant parameters were (GA)(max)= 541 g/L, (SOR)(max)=552 g/L, W(GA)=4.8h, W(SOR)=4.9h, upsilon(GA)=112.7 g/L. and upsilon(SOR)=112.7 g/L.

Frutose, sorbitol e glicose em sangue de mãe, cordão umbilical e recém-nascido de termo com 48 horas de vida / Blood fructose, sorbitol and glucose in mothers, cord blood and in 48-hour-old

Introdução: a frutose é um açúcar derivado da glicose pela via do sorbitol presente em placentas de animais ungulados. Em humanos existem poucos relatos sobre a produção de frutose e de polióis pela unidade feto-placentária. Objetivo: determinar a relação entre os níveis sanguíneos de frutose, sorbitol e glicose em mães, em veia de cordão umbilical e em recém-nascidos de termo em aleitamento materno exclusivo. Métodos: foram coletadas amostras de sangue de 26 mães, imediatamente após o parto, da veia umbilical a 2 cm da placenta e do recém-nascido de termo com peso adequado, 30 minutos após o início da mamada. Frutose, sorbitol e glicose foram determinados por cromatografia líquida de alta resolução (HPLC). A análise estatística foi efetuada por análise de variância de Friedman e por coeficiente de correlação de Spearmann. Resultados: As concentrações de frutose no recém-nascido (105,1 mais ou menos 43,8 micro moI/L) foram superiores às do cordão (77,24 mais ou menos 35,3 micro moI/L) e ambas superiores às maternas (56,04 mais ou menos 21 ,8 micro moI/L) p igual 0,01. Quanto às concentrações de sorbitol, estas foram significantemente mais elevadas no cordão (71,1 masi ou menos 29,6 micro moI/L) em relação à mãe (43,19 mais ou menos 17,8 micro moI/L) e ao recém-nascido (42,67 mais ou menos 22,2 micro moI/L), p igual 0,01 sendo que ambas não diferiram entre si. Quanto às concentrações de glicose, os níveis maternos (4,82 mais ou menos 0,81 mmol/L) foram significantemente maiores que as dos cordões (3,57 mais ou menos 0,72 mmol/L) e dos recém-nascidos (3,04 mais ou menos 0,56 mmol/L) e os níveis dos recém­ nascidos foram inferiores aos dos cordões (p igual 0,01). Observou-se correlação significante entre mãe e cordão para a glicose (r igual 0,62; p menor que 0,0001), não sendo observadas correlações significantes para a frutose e o sorbitol.