Phenolics of the Inner Bark of Silver Birch: Characterization and Intraspecific Variation

This thesis describes work related to the in-depth characterization of the phenolic compounds of silver birch (Betula pendula) inner bark. Phenolic compounds are the most ubiquitous class of plant secondary compounds. The unifying feature of this structurally diverse group is an aromatic ring containing at least one hydroxyl group. Due to the structural diversity, phenolics have various roles in the plant defense against biotic and abiotic stresses. In addition, they can confer several health-promoting properties to humans. Furthermore, the structural diversity of this class of compounds causes challenges for their analysis. The study species in the present work, silver birch, is economically the most important hard wood species in northern Europe. Its inner bark contains a high level of phenolic compounds and it has shown one of the strongest antioxidant activities among 92 Finnish plant materials. The literature review surveys the diversity and organ specific distribution of phenolic compounds in silver birch as well as the proposed ecological functions of phenolic compounds in nature. In addition, the basis for the characterization of phenolics by mass spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR), and circular dichroism spectroscopy (CD) are reviewed. The objective of the experimental work was to extract, purify, characterize, and quantify the inner bark phenolic compounds. Overall 36 compounds were characterized by MS and ultraviolet spectroscopy (UV). 24 compounds were isolated and their structures confirmed by NMR and CD spectroscopy. Five novel natural compounds were identified. Special emphasis was placed on the establishment of a method for the characterization of proanthocyanidins (PAs). Hydrophilic interaction liquid chromatography (HILIC) was utilized because of its high resolution power and predictable elution order of oligomeric and polymeric PAs according to an increasing degree of polymerization. The combination of HILIC and high-resolution MS detection allowed the identification of procyanidin (PC) polymers up to the degree of polymerization of 22. In addition, a series of oligomeric and polymeric PC monoxylosides were observed for the first time in nature. Season and genotype influenced the quantities of the main inner bark phenolics, yet qualitative differences were not observed. However, manual wounding of the inner bark induced the production of ellagitannins (ETs) in the wounded tissues, i.e. callus. Since ETs were not detected in the intact inner bark, this finding may reflect the capacity of silver birch to exploit ellagitannins in its defense.

Effect of nitrate and ammonium on the growth and protein concentration of Microcystis viridis Lemmermann (Cyanobacteria)

Cyanobacteria are a very important group in aquatic systems, particularly in eutrophic waters. Therefore studies about their success in the environment are essential. Many hypotheses have tried to explain the dominance of Cyanobacteria, and several emphasized the importance of various nitrogen sources for the success of the group. In this study, we measured the effect of ammonium and nitrate on the growth and protein concentration of Microcystis viridis (Cyanobacteria). This species is well-known because bloom formation in eutrophic waters. The study was carried out, in experimental batch cultures, using the WC medium with different nitrogen sources: ammonium, nitrate, ammonium + nitrate (50% ammonium + 50% nitrate) and ammonium at different concentrations (to test for possible NH4+ toxicity). Protein, ammonium and nitrate concentrations were measured at end of each experiment, whereas samples for cell counts were taken daily. Results showed that Microcystis viridis grew faster with ammonium (µ = 0.393 day-1) than with nitrate (µ = 0.263 day-1) and ammonium + nitrate (µ = 0.325 day-1). This pattern is explained by the metabolism of ammonium that presents higher uptake and assimilation rates than nitrate. Maximum cell concentration, however, was higher in the ammonium + nitrate treatment, followed by nitrate treatment. Higher protein concentration were observed in the treatment with nitrate. In the ammonium toxicity test, no difference between the control and NH4+ at 50% was found. Thus, the ammonium concentrations used in these experiments were not toxic. Our results suggest that Cyanobacteria is able to grow on both nitrogen sources even if ammonium may allow faster growth and bloom development.

Assessment of silver-stained AFLP markers for studying DNA polymorphism in proso millet (Panicum miliaceum L.)

Proso millet (Panicum miliaceum L.) is a serious weed in North America. A high number of wild proso millet biotypes are known but the genetic basis of its phenotypic variation is poorly understood. In the present study, a non-radioactive silver staining method for PCR-Amplified Fragment Length Polymorphism (AFLP) was evaluated for studying genetic polymorphism in American proso millet biotypes. Twelve biotypes and eight primer combinations with two/three and three/three selective nucleotides were used. Pair of primers with two/three selective nucleotides produced the highest number of amplified DNA fragments, while pair of primers with three/three selective nucleotides were more effective for revealing more polymorphic DNA fragments. The two better primer combinations were EcoR-AAC/Mse-CTT and EcoR-ACT/Mse-CAA with seven and eleven polymorphic DNA fragments, respectively. In a total of 450 amplified fragments, at least 339 appeared well separated in a silver stained acrylamide gel and 39 polymorphic DNA bands were scored. The level of polymorphic DNA (11.5%) using only eight pairs of primers were effective for grouping proso millet biotypes in two clusters but insufficient for separating hybrid biotypes from wild and crop. Nevertheless, the present result indicates that silver stained AFLP markers could be a cheap and important tool for studying genetic relationships in proso millet.

Characterization of nitrate reductase activity in vitro in Gracilaria caudata J. Agardh (Rhodophyta, Gracilariales)

The marine red alga Gracilaria caudata J. Agardh has been used in Brazil for agar extraction, mainly in the northeast region of the country. Nitrogen availability is the most important abiotic factor in seawater that limits the growth of seaweeds. The enzyme nitrate reductase (NR) is the key regulatory point in the nitrogen assimilation in photosynthetic organisms. This study describes an in vitro assay, characterizing the enzymatic activity of NR in terms of kinetic constants and stability, its oscillation during the day and glucose effect on NR modulation. Maximal peaks of NR activity were recorded at 20 ºC and pH 8.0. The enzymatic stability in crude extracts stored at 3 ± 1 ºC decreased significantly after 48 hours. Apparent Michaelis-Menten constants (K M) for NADH and nitrate were 22 µM and 3.95 mM, respectively. Gracilaria caudata NR activity showed an oscillation under light:dark photoperiod (14:10 hours LD) with 3-fold higher activity during the light phase, peaking after 10 hours of light. Under optimal assay conditions, the maximal activity was 92.9 10-3 U g-1. The addition of glucose induced the enzymatic activity during the light and dark phase, evidencing a possible modulation of this enzyme by the photosynthesis. This relationship can be explained by the need of carbon skeletons, produced by the photosynthetic process, to incorporate the intermediary metabolites of nitrate assimilatory pathway, avoiding the toxic intracellular accumulation of nitrite and ammonium. The optimization of enzymatic assay protocols for NR is essential to establish appropriate conditions to study nutritional behaviour, compare different taxonomic groups and to understand its regulatory mechanism.

TRAP-silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines

Measurement of telomerase activity in clinically obtained tumor samples may provide important information for use as both a diagnostic marker and a prognostic indicator for patient outcome. In order to evaluate telomerase activity in tumor tissue without radiolabeling the product, we developed a simple telomeric repeat amplification protocol-silver-staining assay that is less time-consuming, is safe and requires minimal equipment. In addition, we determined the sensitivity of the silver-staining method by using extracts of telomerase-positive thyroid carcinoma cell lines which were serially diluted from 5,000 to 10 cells. Telomerase activity was also assayed in 19 thyroid tumors, 2 normal controls and 27 bone marrow aspirates. The results indicate that the technique permits the detection of telomerase activity from 5000 to as few as 10 cells. We propose that it could be immediately applicable in many laboratories due to the minimal amount of equipment required.

Simulating the dynamics of silver market using computational market dynamics

Traditional econometric approaches in modeling the dynamics of equity and commodity markets, have, made great progress in the past decades. However, they assume rationality among the economic agents and and do not capture the dynamics that produce extreme events (black swans), due to deviation from the rationality assumption. The purpose of this study is to simulate the dynamics of silver markets by using the novel computational market dynamics approach. To this end, the daily data from the period of 1st March 2000 to 1st March 2013 of closing prices of spot silver prices has been simulated with the Jabłonska-Capasso-Morale(JCM) model. The Maximum Likelihood approach has been employed to calibrate the acquired data with JCM. Statistical analysis of the simulated series with respect to the actual one has been conducted to evaluate model performance. The model captures the animal spirits dynamics present in the data under evaluation well.

Correlation between total nitrite/nitrate concentrations and monoamine oxidase (types A and B) and semicarbazide-sensitive amine oxidase enzymatic activities in human mesenteric arteries from non-diabetic and type 2 diabetic patients

The aim of this study was to determine the correlation between total nitrite/nitrate concentrations (NOx) and the kinetic parameters of monoamine oxidase enzymes (MAO-A and MAO-B) and semicarbazide-sensitive amine oxidase (SSAO) in human mesenteric arteries. Arteries were from non-diabetic and type 2 diabetic patients with sigmoid or rectum carcinoma for whom surgery was the first option and who were not exposed to neo-adjuvant therapy. Segments of human inferior mesenteric arteries from non-diabetic (61.1 ± 8.9 years old, 7 males and 5 females, N = 12) and type 2 diabetic patients (65.8 ± 6.2 years old, 8 males and 4 females, N = 12) were used to determine NOx concentrations and the kinetic parameters of MAO-A, MAO-B and SSAO by the Griess reaction and by radiochemical assay, respectively. The NOx concentrations in arteries from diabetic patients did not differ significantly from those of the non-diabetic group (10.28 ± 4.61 vs 10.71 ± 4.32 nmol/mg protein, respectively). In the non-diabetic group, there was a positive correlation between NOx concentrations and MAO-B parameters: Km (r = 0.612, P = 0.034) and Vmax (r = 0.593, P = 0.042), and a negative correlation with the SSAO parameters: Km (r = -0.625, P = 0.029) and Vmax (r = -0.754, P = 0.005). However, in the diabetic group no correlation was found between NOx concentrations and the three kinetic parameters of the enzymes. These results suggest an important function of sympathetic nerves and vascular NOx concentrations in arteries of non-diabetic patients. Thus, these results confirm the importance of a balance between oxidants and antioxidants in the maintenance of vascular homeostasis to prevent oxidative stress.

Sedative and anesthetic activities of the essential oils of Hyptis mutabilis (Rich.) Briq. and their isolated components in silver catfish (Rhamdia quelen)

This study evaluated the sedative and anesthetic effects of the essential oils (EO) of Hyptis mutabilis (Rich.) Briq. and their isolated components on silver catfish (Rhamdia quelen). Quantitative chemical differences between the EOs obtained from leaves and inflorescences were verified, and a new chemotype rich in globulol was described. Although there were no significant differences in the time of induction for sedation and anesthesia between the EOs, only the leaf EO at 344 mg/L anesthetized all fish without side effects. Fractionation of the leaf EO was carried out by column chromatography. The isolated compounds [(+)-1-terpinen-4-ol and (-)-globulol] showed different activity from that detected for the leaf EO in proportional concentrations and similar sedation to a eugenol control at 10 mg/L. However, fish exposed to 1-terpinen-4-ol (3 and 10 mg/L) did not remain sedated for 30 min. Anesthesia was obtained with 83-190 mg/L globulol, but animals showed loss of mucus during induction and mortality at these concentrations. Synergism of the depressor effects was detected with the association of globulol and benzodiazepine (BDZ), compared with either drug alone. Fish exposed to BDZ or globulol+BDZ association showed faster recovery from anesthesia in water containing flumazenil, but the same did not occur with globulol. In conclusion, the use of globulol in aquaculture procedures should be considered only at sedative concentrations of 10 and 20 mg/L, and its mechanism of action seems not to involve the GABAA-BDZ system.

Increased immunoglobulin production in silver catfish (Rhamdia quelen) exposed to agrichemicals

Fish vaccination has been increasingly exploited as a tool to control pathogen infection. The production of immunoglobulin following vaccination might be affected by several factors such as management procedures, water temperature, and the presence of xenobiotics. In the present study, we aimed to investigate the kinetics of immunoglobulin production in silver catfish (Rhamdia quelen) inoculated with inactivated Aeromonas hydrophila and kept at two different water temperatures (17.4±0.4° or 21.3±0.3°C). The effect of a second antigen inoculation and exposure of fish to sublethal concentrations of the herbicides atrazine and glyphosate at 10% of the lethal concentration (LC50-96h) on specific serum antibodies were also investigated. Antibodies to A. hydrophila were detected as early as 7 days post-inoculation and increased steadily up to 35 days. The kinetics of antibody production were similar in fish kept at 17.4±0.4° and 21.3±0.3°C, and reinoculation of antigen at 21 days after priming failed to increase specific antibody levels. Intriguingly, we found that, in fish exposed to atrazine and glyphosate, the secretion of specific antibodies was higher than in non-exposed inoculated fish. These findings are important for the design of vaccines and vaccination strategies in Neotropical fish species. However, because atrazine and glyphosate are widespread contaminants of soil and water, their immune-stimulating effect could be harmful, in that fish living in herbicide-contaminated water might have increased concentrations of nonspecific antibodies that could mediate tissue injury.

27-8-20, Circuit de la Sarthe, Thomas sur Silver Hawk V : [photographie de presse] / [Agence Rol]

Référence bibliographique : Rol, 60761