Interaction of the Fungal Metabolite Harzianic Acid with Rare-Earth Cations (Pr3+, Eu3+, Ho3+, Tm3+).

Rare-earth elements (REEs) are in all respect a class of new contaminants that may have toxic effects on organisms and microorganisms and information on their interactions with natural ligands should be of value to predict and control their diffusion in natural environments. In the current study, we investigate interactions of tripositive cations of praseodymium, europium, holmium, and thulium with harzianic acid (H2L), a secondary metabolite produced by selected strains of fungi belonging to the Trichoderma genus. We applied the same techniques and workflow previously employed in an analogous study concerning lanthanum, neodymium, samarium, and gadolinium tripositive cations. Therefore, in the current study, HPLC-ESI-HRMS experiments, circular dichroism (CD), and UV-Vis spectrophotometric absorption data, as well as accurate pH measurements, were applied to characterize bonding interactions between harzianic acid and Pr3+, Eu3+, Ho3+, and Tm3+ cations. Problems connected to the low solubility of harzianic acid in water were overcome by employing a 0.1 M NaClO4/(CH3OH + H2O 50/50 w/w) mixed solvent. For Pr3+, Ho3+, and Tm3+, only the mono complexes PrL+, HoL+, and TmL+ were detected and their formation constant determined. Eu3+ forms almost exclusively the bis complex EuL2- for which the corresponding formation constant is reported; under our experimental conditions, the mono complex EuL+ is irrelevant. Combining the results of the present and previous studies, a picture of interactions of harzianic acid with rare-earth cations extending over 8 of the 17 REEs can be composed. In order to complement chemical information with toxicological information, a battery of bioassays was applied to evaluate the effects of praseodymium, europium, holmium, and thulium tripositive cations on a suite of bioindicators including Aliivibrio fischeri (Gram-negative bacterium), Raphidocelis subcapitata (green alga), and Daphnia magna (microcrustacean), and median effective concentration (EC50) values of Pr3+, Eu3+, Ho3+, and Tm3+ for the tested species were assessed.

Interaction of the Fungal Metabolite Harzianic Acid with Rare-Earth Cations (La3+, Nd3+, Sm3+, Gd3+).

Rare-earth elements are emerging contaminants of soil and water bodies which destiny in the environment and effects on organisms is modulated by their interactions with natural ligands produced by bacteria, fungi and plants. Within this framework, coordination by harzianic acid (H2L), a Trichoderma secondary metabolite, of a selection of tripositive rare-earth cations Ln3+ (Ln3+ = La3+, Nd3+, Sm3+, and Gd3+) was investigated at 25 °C, and in a CH3OH/0.1 M NaClO4 (50/50 w/w) solvent, using mass spectrometry, circular dichroism, UV-Vis spectrophotometry, and pH measurements. Experimental data can be satisfactorily explained by assuming, for all investigated cations, the formation of a mono-complex (LnL+) and a bis-complex (LnL2-). Differences were found between the formation constants of complexes of different Ln3+ cations, which can be correlated with ionic radius. Since gadolinium is the element that raises the most concern among lanthanide elements, its effects on organisms at different levels of biological organization were explored, in the presence and absence of harzianic acid. Results of ecotoxicological tests suggest that harzianic acid can decrease gadolinium biotoxicity, presumably because of complex formation with Gd3+.

Solubility of folic acid and protonation of folate in NaCl at different concentrations, even in physiological solution.

The solubility of folic acid was determined at 25 °C in 1.00 mol dm-3 and in 0.15 mol dm-3 NaCl (physiological solution) spectrophotometrically by measuring the absorbance of saturated solution at different hydrogen ion concentrations. Five protonation constants of folate were determined both from the dependence of the solubility on the hydrogen ion concentration as well as from potentiometric titrations carried out in the presence of solid folic acid and in alkaline solution, in which folate is relatively soluble. Corresponding to the protonation constants, nuclear magnetic resonance and florescence spectra were also obtained at different hydrogen ion concentrations to determine the protonation positions in acid, neutral and alkaline solutions. An approach through circular dichroism was also applied to study the eventual polymerization of folate in alkaline solution.

Bivalent Metal-Chelating Properties of Harzianic Acid Produced by Trichoderma pleuroticola Associated to the Gastropod Melarhaphe neritoides.

Harzianic acid is a secondary metabolite of Trichoderma, structurally belonging to the dienyltetramic acid subgroup of the tetramic acids. Biological activities of harzianic acid are of great interest for its antimicrobial and plant growth-promoting activities, which might be related to its chelating properties. In the present work harzianic acid, isolated from cultures of a strain of Trichoderma pleuroticola associated to the gastropod Melarhaphe neritoides, was studied as a complexant agent of a number of biologically relevant transition metals (i.e., Zn2+, Fe2+, Cu2+, and Mn2+), using UV-VIS, potentiometry, MS and NMR techniques. Our findings show the coordination capacity of harzianic acid toward the above cations through the formation of neutral or charged complexes in a variable ratio depending on the metal and pH conditions.

The Issue of Misidentification of Kojic Acid with Flufuran in Aspergillus flavus.

In the course of investigations on the complex phenomenon of bee decline, Aspergillus flavus was isolated from the haemocoel of worker bees. Observations on the metabolomic profile of this strain showed kojic acid to be the dominant product in cultures on Czapek-Dox broth. However, an accurate review of papers documenting secondary metabolite production in A. flavus also showed that an isomer of kojic acid, identified as 5-(hydroxymethyl)-furan-3-carboxylic acid and named flufuran is reported from this species. The spectroscopic data of kojic acid were almost identical to those reported in the literature for flufuran. This motivated a comparative study of commercial kojic acid and 5-(hydroxymethyl)-furan-3-carboxylic acid, highlighting some differences, for example in the 13C-NMR and UV spectra for the two compounds, indicating that misidentification of the kojic acid as 5-(hydroxymethyl)-furan-3-carboxylic acid has occurred in the past.

Silibinin phosphodiester glyco-conjugates: Synthesis, redox behaviour and biological investigations.

New silibinin phosphodiester glyco-conjugates were synthesized by efficient phosphoramidite chemistry and were fully characterized by 2D-NMR. A wide-ranging study focused on the determination of their pKa and E° values as well as on their radical scavenging activities by different assays (DPPH, ABTS+ and HRSA) was conducted. The new glyco-conjugates are more water-soluble than silibinin, and their radical scavenging activities are higher than those of silibinin. The conjugation therefore improves both the water solubilities and antioxidant activities of the flavonolignan moieties. The serum stability was evaluated under physiological conditions, and the glyco-conjugates degraded with half-lives of 40-70 h, making them useful in pro-drug approaches. We started by treating androgen-dependent prostate cancer (PCa) LNCaP cells and then expanded our studies to androgen-independent PCa PC3 and DU145 cells. In most cases, the new derivatives significantly reduced both total and live cell numbers, albeit at different levels. Anti-HIV activities were evaluated and the glucosamine-phosphate silibinin derivative showed higher activity (IC50 = 73 µM) than silibinin.

Biomonitoring of Airborne Microplastic Deposition in Semi-Natural and Rural Sites Using the Moss Hypnum cupressiforme.

We show that the native moss Hypnum cupressiforme can be used as a biomonitor of atmospheric microplastics (MPs). The moss was collected in seven semi-natural and rural sites in Campania (southern Italy) and was analyzed for the presence of MPs, according to standard protocols. Moss samples from all sites accumulated MPs, with fibers representing the largest fraction of plastic debris. Higher numbers of MPs and longer fibers were recorded in moss samples from sites closer to urbanized areas, likely as the results of a continuous flux from sources. The MP size class distribution showed that small size classes characterized sites having a lower level of MP deposition and a high altitude above sea level.

Harzianic Acid Activity against Staphylococcus aureus and Its Role in Calcium Regulation.

Staphylococcus aureus is a Gram-positive bacterium, which can be found, as a commensal microorganism, on the skin surface or in the nasal mucosa of the human population. However, S. aureus may become pathogenic and cause severe infections, especially in hospitalized patients. As an opportunistic pathogen, in fact, S. aureus interferes with the host Ca2+ signaling, favoring the spread of the infection and tissue destruction. The identification of novel strategies to restore calcium homeostasis and prevent the associated clinical outcomes is an emerging challenge. Here, we investigate whether harzianic acid, a bioactive metabolite derived from fungi of the genus Trichoderma, could control S. aureus-induced Ca2+ movements. First, we show the capability of harzianic acid to complex calcium divalent cations, using mass spectrometric, potentiometric, spectrophotometric, and nuclear magnetic resonance techniques. Then, we demonstrate that harzianic acid significantly modulates Ca2+ increase in HaCaT (human keratinocytes) cells incubated with S. aureus. In conclusion, this study suggests harzianic acid as a promising therapeutical alternative against diseases associated with Ca2+ homeostasis alteration.

Facing metal stress by multiple strategies: morphophysiological responses of cardoon (Cynara cardunculus L.) grown in hydroponics.

The contamination of environments by heavy metals has become an urgent issue causing undesirable accumulations and severe damages to agricultural crops, especially cadmium and lead which are among the most widespread and dangerous metal pollutants worldwide. The selection of proper species is a crucial step in many plant-based restoration approaches; therefore, the aim of the present work was to check for early morphophysiological responsive traits in three cultivars of Cynara cardunculus (Sardo, Siciliano, and Spagnolo), helping to select the best performing cultivar for phytoremediation. For all three tested cultivars, our results indicate that cardoon displays some morphophysiological traits to face Cd and Pb pollution, particularly at the root morphology level, element uptake ability, and photosynthetic pigment content. Other traits show instead a cultivar-specific behavior; in fact, stomata plasticity, photosynthetic pattern, and antioxidant power provide different responses, but only Spagnolo cv. achieves a successful strategy attaining a real resilience to metal stress. The capacity of Spagnolo plants to modify leaf structural and physiological traits under heavy metal contamination to maintain high photosynthetic efficiency should be considered an elective trait for its use in contaminated environments.

Coordination Properties of the Fungal Metabolite Harzianic Acid toward Toxic Heavy Metals.

Some Trichoderma strains are known for their capacity to produce harzianic acid, a metabolite belonging to the tetramic acid derivatives. Harzianic acid has interesting biological properties, such as antimicrobial activities against phytopathogenic fungi and promotion of plant growth. It also possesses remarkable chemical properties, including the chelating properties toward essential transition metals, which might be related to the biological activities. Increasing knowledge on chelating properties might be relevant for understanding the various beneficial effects of harzianic acid in the interaction between the producer fungi and plants. In this work, the coordination capacity of harzianic acid was studied to evaluate the formation and stability of complexes formed with toxic heavy metals (i.e., Cd2+, Co2+, Ni2+, and Pb2+), which might have a crucial role in the tolerance of plants growing in metal-contaminated soils and in abiotic stress.

Synthesis of New Tyrosol-Based Phosphodiester Derivatives: Effect on Amyloid ß Aggregation and Metal Chelation Ability.

Alzheimer's disease (AD) is a multifactorial pathology that requires multifaceted agents able to address its peculiar nature. Increasing evidence has shown that aggregation of amyloid ß (Aß) and oxidative stress are strictly interconnected, and their modulation might have a positive and synergic effect in contrasting AD-related impairments. Herein, a new and efficient fragment-based approach towards tyrosol phosphodiester derivatives (TPDs) has been developed starting from suitable tyrosol building blocks and exploiting the well-established phosphoramidite chemistry. The antioxidant activity of new TPDs has been tested as well as their ability to inhibit Aß protein aggregation. In addition, their metal chelating ability has been evaluated as a possible strategy to develop new natural-based entities for the prevention or therapy of AD. Interestingly, TPDs containing a catechol moiety have demonstrated highly promising activity in inhibiting the aggregation of Aß40 and a strong ability to chelate biometals such as CuII and ZnII .

Behavior of glutathione as ligand of lead (II).

The presence and mobilization of toxic metal cations represents under many aspects a current and important problem in the environmental field. In this research, as cation lead (II) ion was studied. The formation of complexes between glutathione and lead (II) was studied at 25 °C and in 1.00 M NaCl as ionic medium by means of measurements of electromotive force (e.m.f.) of cells containing glass and lead amalgam electrodes. In the same experimental conditions, the protonation constants of glutathione were determined potentiometrically, using a cell containing the glass electrode. In the same experimental conditions, by considering glutathione (GSH) completely deprotonated, four protonation constants were determined. Potentiometric data could be explained by assuming the formation of 1:1 complexes between GSH and Pb2+ and with the participation of hydrogen ions. The stability constants of the assumed complexes were determined. The 1:1 ratio between GSH and lead (II) was confirmed by spectrophotometric investigations. Measurements by Infrared Rays (IR) and protonic Nuclear Magnetic Resonance (1H NMR) provide information on the structure of the found complexes.

Coordination of a bis-histidine-oligopeptide to Re(i) and Ga(iii) in aqueous solution.

The utilization of isotopes of transition metals for the development of novel therapeutic or diagnostic compounds is limited by the fact that they must be stabilized by chelating systems in coordination complexes. Important roles in the targeting approach are played by the tricarbonyl complexes of Technetium(i) and Rhenium(i) because they can be readily conjugated to biomolecules to form stable probes. Additionally, 67Ga and 68Ga isotopes of gallium are considered an obvious alternative to 99mTc (M. D. Bartholomä, A. S. Louie, J. F. Valliant and J. Zubieta, Chem. Rev., 2010, 110, 2903-2920) for SPECT and PET applications. We have previously reported the characterization of the peptide CCK8 decorated with a bis-histidine-based chelator (pHis2) labeled with 99mTc-tricarbonyl. In order to study the molecular properties of the histidine-based chelator pHis2, we here present the characterization in solution of its complexes with the metals Re(i) and Ga(iii) using potentiometry and NMR. We detail the solution equilibria reporting pHis2 acid-base behavior, the coordination properties of pHis2 toward fac-[Re(H2O)3(CO)3]+ and Ga(iii) and the atomic details of the formed complexes. Interestingly, two different metal coordination modes were found highlighting the plasticity of this bifunctional chelator.

Protolytic behavior of hydroxylated Pyrex glass surfaces in NaCl media.

Adsorption of hydrogen ions from aqueous NaCl solutions at the Pyrex glass-water interface was investigated by acid-base titration (glass electrode) at 25 degrees C and at the ionic strengths 0.010, 0.030, 0.10, 1.0, and 3.0 mol dm(-3). The pH values ranged from 2 to 7. The Pyrex samples had a specific surface area of 19.2x10(3) m(2)kg(-1) and a porous structure (pores 2.4 nm thick, 280 nm long). The reactions were found to be extremely slow but showed good reversibility. The potentiometric data, due to the small effect of ionic strength on the equilibria, were fitted with a simple nonelectrostatic model based on strong specific interactions of medium ions with deprotonated silanol, >SiO(-), and boranol, >BO(-), as well as with protonated sites. The acid-base properties are described by the reactions and equilibrium constants at the infinite dilution reference state: >SiONa + H(+) <==> >SiOHNa(+), logbeta110Si=3.1+/-0.2; >SiONa + 2H(+) + Cl(-) <==> >SiOH(2)Cl + Na(+), logbeta201Si=6.75+/-0.15; >SiONa + H(+) <==> >SiOH + Na(+), logbeta100Si=1.8+/-0.2, >BONa + H(+) <==>>BOH + Na(+), logbeta100B=6.4+/-0.2; >BONa + H(+) <==> >BOHNa(+), logbeta110B=6.6+/-0.2; >BONa + 2H(+) <==> >BOH(+)(2) + Na(+), logbeta200B=11.56+/-0.15.

Stability constants of iron(II) sulfate complexes.

The complex formation equilibria between iron(II) and sulfate ions have been studied at 25 degrees C in 3 M NaClO4 ionic medium by measuring with a glass electrode the competition of Fe2+ and H+ ions for the sulfate ion. The concentrations of the metal and of the ligand were varied in the ranges 0.01 to 0.125 and 0.01 to 0.250 M, respectively. The analytical concentration of strong acid was chosen to be 0.01 or 0.03 M. The potentials of the glass electrode, corrected for the effect of replacement of medium ions with reagent species, have been interpreted with the equilibria [formula: see text] Stability constants valid in the infinite dilution reference state, logK zero = 1.98 +/- 0.16, log beta 1 zero = 2.1(5) +/- 0.2 and log beta 2 = 2.5 +/- 0.2, have been estimated by assuming the validity of the specific interaction theory.

On the complex formation equilibria between iron (III) and sulfate ions.

The equilibria have been investigated at 25 degrees C in 3 M NaClO4 using potentiometry, glass and redox Fe3+/Fe2+ half-cells, and UV optical absorptiometry. The concentration of the reagents was chosen in the intervals: 10(-4) < or = [Fe(III)] < or = 5.10(-3) M, 0.01 < or = [SO4(2-)]tot < or = 0.65 M. The value of [H+] was kept at 0.1 M or more to reduce the hydrolysis of the Fe3+ ion to less than 1%. Auxiliary constants, corresponding to the formation of Fe(II)-sulfate complexes and to the association of H+ with SO4(2-) ions, were taken from previous determinations. The experimental data could be explained with the equilibria [formula: see text] Equilibrium constants at infinite dilution, log beta 101 degrees = 3.82 +/- 0.17, log beta 102 degrees = 5.75 +/- 0.17 and log beta 111 degrees = 3.68 +/- 0.35, have been evaluated by applying the specific interaction theory.

Hydroxo sulfate complexes of iron (III) in solution.

The ternary Fe (III)-OH(-)-SO4(2-) complexes have been investigated at 25 degrees C in 3 M NaClO4 by potentiometric titration with glass electrode. The metal and sulfate concentrations ranged from 2.5 x 10(-3) to 0.03 M and from 5.10(-3) to 0.060 M, respectively. [H+] was decreased from 0.05 M to incipient precipitation of basic sulfate which occured at log[H+] between -2.3 and -2.5 depending on the concentration of the metal. For the interpretation of the data stability constants of HSO4(-), of binary hydroxo complexes (FeOH2+, Fe(OH)2+, Fe2(OH)2(4+), Fe3(OH)4(5+), Fe3(OH)5(4+)) and of sulfate complexes (FeSO4+, FeHSO4(2+), Fe(SO4)2-) were assumed from independent sources. The data are consistent with the presence of FeOHSO4, log beta 1-11 = -0.49 +/- 0.03. Equilibrium constants are defined as beta pqr for pFe3+ +qH+ +rSO4(2-) [symbol: see text] FepHq(SO4)r3p+q-2r. No substantial better fit could be found by adding a second mixed complex. Only a slightly smaller agreement factor resulted introducing as minor ternary complex Fe3(OH)6(SO4)3(3-) with log beta 3-63 = -5.8 +/- 0.5. Its evidence, however, cannot be considered conclusive.

The acidic constants of 2-hydroxybenzohydroxamic acid in NaClO4 solutions at 25 degrees C.

The protolysis equilibria of 2-hydroxybenzohydroxamic acid, H2SAX, have been studied at 25 degrees C in different ionic media by potentiometric titration with a glass electrode. The media were 0.513, 1.05, 2.21 and 3.5 mol/kg NaClO4. The constants beta(-p)(H2SAX<==>H(2-p)SAX(-p)+pH+), combined with salting effects of NaClO4 on H2SAX deduced from solubility determinations, were processed by the specific interaction theory, SIT, to give equilibrium constants at infinite dilution, log beta(-1)(o) = -7.655 +/- 0.013 and log beta(-2)(o) = -17.94 +/- 0.04, as well as specific interaction coefficients b(HSAX-,Na+) = 0.12 +/- 0.01 and b(SAX2-,Na+) = 0.17 +/- 0.02, molal(-1).

On the complex formation equilibria between dioxouranium(VI) and sulfate ions.

The complexation equilibria between UO2(2+) and SO4(2-) ions have been studied at 25 degrees C in the ionic medium 3 M NaClO4 by potentiometry, by spectrophotometry and by solubility measurements of UO2(IO3)2. The potentiometric investigation was carried out with the Hg-Hg2SO4(s)-SO4(2-) half-cell and glass electrode in the sulfate concentration range 0.005 to 0.07 M. The optical absorbances in the UV-visible region and the solubility data cover the ligand concentration range 0.005 to 0.3 M. The data could be explained by assuming the complexes and equilibrium constants [Table: see text]. The constants in the infinite dilution reference state, log beta1o = 3.08 +/- 0.15 and log beta2o = 4.28 +/- 0.15, estimated by assuming the validity of the specific interaction theory, are practically coincident with literature data.

On the hydrolysis of the dioxouranium(VI) ion in sulfate solutions.

The formation of ternary UO2(2+)-(OH-)-SO4(2-) complexes has been studied at 25 degrees C in 3 M NaClO4 ionic medium by measurements with a glass electrode. The solutions had uranium concentrations between 0.3 and 30 mM, sulfate between 20 and 200 mM, and 1.66 < or = [SO4(2-)]/[U(VI)] < or = 300. The hydrogen ion concentration ranged from 10(-3) M to incipient precipitation of basic sulfates. This occurred, depending on the metal concentration, at [H+] between 10(-4) and 10(-5.3) M. In the interpretation of the data the stabilities of binary complexes were assumed from independent sources. The data could be explained with the mixed complexes and equilibria (beta(pqr)(3sigma) refers to pUO2(2+) + qH2O + rSO4(2-) <==> (UO2)p(OH)q(SO4)r(2p-q-2r) + qH+): logbeta222 = -2.94 +/- 0.03, logbeta341 = -9.82 +/- 0.06, logbeta211 = -0.30 +/- 0.09, logbeta212 = 1.09 +/- 0.09, logbeta351 = -15.04 +/- 0.09 and logbeta462 = -14.40 +/- 0.06. The fit could be improved by including UO2OH+ with logbeta110 = -5.1 +/- 0.1. The identity of the minor species remains, however, an open question.