Identification of ginsenosides in roots of Panax ginseng by HPLC-APCI/MS

A new HPLC-APCI/MS method for the identification of ginsenosides has been developed. The analyses were performed on a reversed-phase C-18 column using a binary eluent (acetonitrile and water) under gradient conditions. Although APCI is a high-temperature evaporative process, HPLC-APCI/MS could effectively identify thermo-labile ginsenosides. The [M-H](-) ions and the thermal degradation ions of ginsenosides could be clearly observed under negative and positive ion conditions, respectively, and these were used to identify the molecular masses, the aglycone structures and the sugar groups of ginsenosides. APCI/MS can provide more explicit information than ESI/MS for identifying and distinguishing ginsenosides. Using the HPLC-APCI/MS method, 35 ginsenosides were identified in Panax ginseng. Copyright (C) 2005 John Wiley & Sons, Ltd.

Effects of High Dissolved Inorganic and Organic Carbon Availability on the Physiology of the Hard Coral Acropora millepora from the Great Barrier Reef

Coral reefs are facing major global and local threats due to climate change-induced increases in dissolved inorganic carbon (DIC) and because of land-derived increases in organic and inorganic nutrients. Recent research revealed that high availability of labile dissolved organic carbon (DOC) negatively affects scleractinian corals. Studies on the interplay of these factors, however, are lacking, but urgently needed to understand coral reef functioning under present and near future conditions. This experimental study investigated the individual and combined effects of ambient and high DIC (pCO2 403 μatm/ pHTotal 8.2 and 996 μatm/pHTotal 7.8) and DOC (added as Glucose 0 and 294 μmol L-1, background DOC concentration of 83 μmol L-1) availability on the physiology (net and gross photosynthesis, respiration, dark and light calcification, and growth) of the scleractinian coral Acropora millepora (Ehrenberg, 1834) from the Great Barrier Reef over a 16 day interval. High DIC availability did not affect photosynthesis, respiration and light calcification, but significantly reduced dark calcification and growth by 50 and 23%, respectively. High DOC availability reduced net and gross photosynthesis by 51% and 39%, respectively, but did not affect respiration. DOC addition did not influence calcification, but significantly increased growth by 42%. Combination of high DIC and high DOC availability did not affect photosynthesis, light calcification, respiration or growth, but significantly decreased dark calcification when compared to both controls and DIC treatments. On the ecosystem level, high DIC concentrations may lead to reduced accretion and growth of reefs dominated by Acropora that under elevated DOC concentrations will likely exhibit reduced primary production rates, ultimately leading to loss of hard substrate and reef erosion. It is therefore important to consider the potential impacts of elevated DOC and DIC simultaneously to assess real world scenarios, as multiple rather than single factors influence key physiological processes in coral reefs.

Arctic microorganisms respond more to elevated UV-B radiation than CO2

David Johnson, Colin D. Campbell, John A. Lee, Terry V. Callaghan and Dylan Gwynn-Jones (2002). Arctic microorganisms respond more to elevated UV-B radiation than CO2. Nature, 416 (6876) pp.82-83 Sponsorship: NERC / EU / Swedish Academy of Sciences RAE2008

Formulações nanobiotecnológicas para vetorização da Curcumina e seus derivados

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Synthesis and reactivity of α-diazo-β-keto sulfoxides: scope and synthetic potential

The research described in this thesis focuses on the design and synthesis of stable α-diazosulfoxides and investigation of their reactivity under a variety of conditions (transition-metal catalysis, thermal, photochemical and microwave) with a particular emphasis on the synthesis of novel heterocyclic compounds with potential biological activity. The exclusive reaction pathway for these α-diazosulfoxides was found to be hetero-Wolff rearrangement to give α-oxosulfine intermediates. In the first chapter, a literature review of sulfines is presented, including a discussion of naturally occurring sulfines, and an overview of the synthesis and reactivity of sulfines. The potential of sulfines in organic synthesis and recent developments in particular are highlighted. The second chapter discusses the synthesis and reactivity of α-diazosulfoxides, building on earlier results in this research group. The synthesis of lactone-based α-diazosulfoxides and, for the first time, ketone-based benzofused and monocyclic α-diazosulfoxides is described. The reactivity of these α-diazosulfoxides is then explored under a variety of conditions, such as transition-metal catalysis, photochemical and microwave, generating labile α-oxosulfine intermediates, which are trapped using amines and dienes, in addition to the spontaneous reaction pathways which occur with α-oxosulfines in the absence of a trap. A new reaction pathway was explored with the lactone based α-oxosulfines, involving reaction with amines to generate novel 3-aminofuran-2(5H)-ones via carbophilic attack, in very good yields. The reactivity of ketone-based α-diazosulfoxides was explored for the first time, and once again, pseudo-Wolff rearrangement to the α-oxosulfines was the exclusive reaction pathway observed. The intermediacy of the α-oxosulfines was confirmed by trapping as cycloadducts, with the stereochemical features dependant on the reaction conditions. In the absence of a diene trap, a number of reaction fates from the α-oxosulfines were observed, including complete sulfinyl extrusion to give indanones, sulfur extrusion to give indanediones, and, to a lesser extent, dimerisation. The indanediones were characterised by trapping as quinoxalines, to enable full characterisation. One of the overriding outcomes of this thesis was the provision of new insights into the behaviour of α-oxosulfines with different transition metal catalysts, and under thermal, microwave and photolysis conditions. A series of 3-aminofuran-2(5H)-ones and benzofused dihydro-2H-thiopyran S-oxides were submitted for anticancer screening at the U.S. National Cancer Institute. A number of these derivatives were identified as hit compounds, with excellent cell growth inhibition. One 3-aminofuran-2(5H)-one derivative has been chosen for further screening. The third chapter details the full experimental procedures, including spectroscopic and analytical data for the compounds prepared during this research. The data for the crystal structures are contained in the attached CD.

Direct and Indirect Effects of Dissolved Organic Matter Source and Concentration on Denitrification in Northern Florida Rivers

Using a natural gradient of dissolved organic carbon (DOC) source and concentration in rivers of northern Florida, we investigated how terrestrially-derived DOC affects denitrification rates in river sediments. Specifically, we examined if the higher concentrations of DOC in blackwater rivers stimulate denitrification, or whether such terrestrially-derived DOC supports lower denitrification rates because (1) it is less labile than DOC from aquatic primary production; whether (2) terrestrial DOC directly inhibits denitrification via biochemical mechanisms; and/or whether (3) terrestrial DOC indirectly inhibits denitrification via reduced light availability to-and thus DOC exudation by-aquatic primary producers. We differentiated among these mechanisms using laboratory denitrification assays that subjected river sediments to factorial amendments of NO3- and dextrose, humic acid dosing, and cross-incubations of sediments and water from different river sources. DOC from terrestrial sources neither depressed nor stimulated denitrification rates, indicating low lability of this DOC but no direct inhibition; humic acid additions similarly did not affect denitrification rates. However, responses to addition of labile C increased with long-term average DOC concentration, which supports the hypothesis that terrestrial DOC indirectly inhibits denitrification via decreased autochthonous production. Observed and future changes in DOC concentration may therefore reduce the ability of inland waterways to remove reactive nitrogen. © 2013 Springer Science+Business Media New York.

The Function and Regulation of Photobodies in Phytochrome Signaling

Light is a critical environmental signal that regulates every phase of the plant life cycle, from germination to floral initiation. Of the many light receptors in the model plant Arabidopsis thaliana, the red- and far-red light-sensing phytochromes (phys) are arguably the best studied, but the earliest events in the phy signaling pathway remain poorly understood. One of the earliest phy signaling events is the translocation of photoactivated phys from the cytoplasm to the nucleus, where they localize to subnuclear foci termed photobodies; in continuous light, photobody localization correlates closely with the light-dependent inhibition of embryonic stem growth. Despite a growing body of evidence supporting the biological significance of photobodies in light signaling, photobodies have also been shown to be dispensable for seedling growth inhibition in continuous light, so their physiological importance remains controversial; additionally, the molecular components that are required for phy localization to photobodies are largely unknown. The overall goal of my dissertation research was to gain insight into the early steps of phy signaling by further defining the role of photobodies in this process and identifying additional intragenic and extragenic requirements for phy localization to photobodies.

Even though the domain structure of phys has been extensively studied, not all of the intramolecular requirements for phy localization to photobodies are known. Previous studies have shown that the entire C-terminus of phys is both necessary and sufficient for their localization to photobodies. However, the importance of the individual subdomains of the C-terminus is still unclear. For example a truncation lacking part of the most C-terminal domain, the histidine kinase-related domain (HKRD), can still localize to small photobodies in the light and behaves like a weak allele. However, a point mutation within the HKRD renders the entire molecule completely inactive. To resolve this discrepancy, I explored the hypothesis that this point mutation might impair the dimerization of the HKRD; dimerization has been shown to occur via the C-terminus of phy and is required for more efficient signaling. I show that this point mutation impairs nuclear localization of phy as well as its subnuclear localization to photobodies. Additionally, yeast-two-hybrid analysis shows that the wild-type HKRD can homodimerize but that the HKRD containing the point mutation fails to dimerize with both itself and with wild-type HKRD. These results demonstrate that dimerization of the HKRD is required for both nuclear and photobody localization of phy.

Studies of seedlings grown in diurnal conditions show that photoactivated phy can persist into darkness to repress seedling growth; a seedling's growth rate is therefore fastest at the end of the night. To test the idea that photobodies could be involved in regulating seedling growth in the dark, I compared the growth of two transgenic Arabidopsis lines, one in which phy can localize to photobodies (PBG), and one in which it cannot (NGB). Despite these differences in photobody morphology, both lines are capable of transducing light signals and inhibiting seedling growth in continuous light. After the transition from red light to darkness, the PBG line was able to repress seedling growth, as well as the accumulation of the growth-promoting, light-labile transcription factor PHYTOCHROME INTERACTING FACTOR 3 (PIF3), for eighteen hours, and this correlated perfectly with the presence of photobodies. Reducing the amount of active phy by either reducing the light intensity or adding a phy-inactivating far-red pulse prior to darkness led to faster accumulation of PIF3 and earlier seedling growth. In contrast, the NGB line accumulated PIF3 even in the light, and seedling growth was only repressed for six hours; this behavior was similar in NGB regardless of the light treatment. These results suggest that photobodies are required for the degradation of PIF3 and for the prolonged stabilization of active phy in darkness. They also support the hypothesis that photobody localization of phys could serve as an instructive cue during the light-to-dark transition, thereby fine-tuning light-dependent responses in darkness.

In addition to determining an intragenic requirement for photobody localization and further exploring the significance of photobodies in phy signaling, I wanted to identify extragenic regulators of photobody localization. A recent study identified one such factor, HEMERA (HMR); hmr mutants do not form large photobodies, and they are tall and albino in the light. To identify other components in the HMR-mediated branch of the phy signaling pathway, I performed a forward genetic screen for suppressors of a weak hmr allele. Surprisingly, the first three mutants isolated from the screen were alleles of the same novel gene, SON OF HEMERA (SOH). The soh mutations rescue all of the phenotypes associated with the weak hmr allele, and they do so in an allele-specific manner, suggesting a direct interaction between SOH and HMR. Null soh alleles, which were isolated in an independent, tall, albino screen, are defective in photobody localization, demonstrating that SOH is an extragenic regulator of phy localization to photobodies that works in the same genetic pathway as HMR.

In this work, I show that dimerization of the HKRD is required for both the nuclear and photobody localization of phy. I also demonstrate a tight correlation between photobody localization and PIF3 degradation, further establishing the significance of photobodies in phy signaling. Finally, I identify a novel gene, SON OF HEMERA, whose product is necessary for phy localization to photobodies in the light, thereby isolating a new extragenic determinant of photobody localization. These results are among the first to focus exclusively on one of the earliest cellular responses to light - photobody localization of phys - and they promise to open up new avenues into the study of a poorly understood facet of the phy signaling pathway.

Early social exposure in wild chimpanzees: mothers with sons are more gregarious than mothers with daughters.

In many mammals, early social experience is critical to developing species-appropriate adult behaviors. Although mother-infant interactions play an undeniably significant role in social development, other individuals in the social milieu may also influence infant outcomes. Additionally, the social skills necessary for adult success may differ between the sexes. In chimpanzees (Pan troglodytes), adult males are more gregarious than females and rely on a suite of competitive and cooperative relationships to obtain access to females. In fission-fusion species, including humans and chimpanzees, subgroup composition is labile and individuals can vary the number of individuals with whom they associate. Thus, mothers in these species have a variety of social options. In this study, we investigated whether wild chimpanzee maternal subgrouping patterns differed based on infant sex. Our results show that mothers of sons were more gregarious than mothers of daughters; differences were especially pronounced during the first 6 mo of life, when infant behavior is unlikely to influence maternal subgrouping. Furthermore, mothers with sons spent significantly more time in parties containing males during the first 6 mo. These early differences foreshadow the well-documented sex differences in adult social behavior, and maternal gregariousness may provide sons with important observational learning experiences and social exposure early in life. The presence of these patterns in chimpanzees raises questions concerning the evolutionary history of differential social exposure and its role in shaping sex-typical behavior in humans.

Fósforo extractable y fósforo orgánico lábil como indicadores de calidad de suelos de Entre Ríos

p.41-46

Aspects of the in vitro bioactivity and antimicrobial properties of Ag+- and Zn2+-exchanged 11 Å tobermorites

11 Å tobermorite, Ca5Si6O16(OH)2 · 4H2O, is a layer lattice ion exchange mineral whose potential as a carrier for Ag+ and Zn2+ ions in antimicrobial, bioactive formulations has not yet been explored. In view of this, the in vitro bioactivity of Ag+- and Zn2+-exchanged 11 Å tobermorites and their bactericidal action against S. aureus and P.aeruginosa are reported. The in vitro bioactivity of the synthetic unsubstituted tobermorite phase was confirmed by the formation of bone-like hydroxycarbonate apatite (HCA) on its surface within 48 h of contact with simulated body fluid. The substitution of labile Ag+ ions into the tobermorite lattice delayed the onset of HCA-formation to 72 h; whereas, the Zn2+-substituted phase failed to elicit an HCA-layer within 14 days. Both Ag+- and Zn2+-exchanged tobermorite phases were found to exhibit marked antimicrobial action against S. aureus and P.aeruginosa, two common pathogens in biomaterial-centred infections.

Ag+- and Zn2+-exchange kinetics and antimicrobial properties of 11 angstrom tobermorites

Ag+- and Zn2+-exchanged zeolites zeolites and clays have been used as coatings and in composites to confer broad-spectrum antimicrobial properties on a range of technical and biomedical materials. 11 angstrom tobermorite is a bioactive layer lattice ion exchanger whose potential as a carrier for Ag+ and Zn2+ ions in antimicrobial formulations has not yet been explored. In view of this, batch Ag+- and Zn2+-exchange kinetics of two structurally distinct synthetic 11 angstrom tobermorites and their subsequent bactericidal action against Staphylococcus aureus and Pseudomonas aeruginosa are reported. During the exchange reactions, Ag+ ions were found to replace labile interlayer cations; whereas, Zn2+ ions also displaced structural Ca2+ ions from the tobermorite lattice. In spite of these different mechanisms, a simple pseudo-second-order model provided a suitable description of both exchange processes (R-2 >= 0.996). The Ag+- and Zn2+-exchanged tobermorite phases exhibited marked bacteriostatic effects against both bacteria, and accordingly, their potential for use as antimicrobial materials for in situ bone tissue regeneration is discussed. (C) 2008 Elsevier Ltd. All rights reserved.

IR/Raman spectroscopy and DFT calculations of cyclic di-amino acid peptides. Part III: comparison of solid state and solution structures of cyclo(L-Ser-L-Ser)

B3-LYP/cc-pVDZ calculations of the gas-phase structure and vibrational spectra of the isolated molecule cyclo(L-Ser-L-Ser), a cyclic di-amino acid peptide (CDAP), were carried out by assuming C-2 symmetry. It is predicted that the minimum-energy structure is a boat conformation for the diketopiperazine (DKP) ring with both L-Beryl side chains being folded slightly above the ring. An additional structure of higher energy (15.16 kJ mol(-1)) has been calculated for a DKP ring with a planar geometry, although in this case two fundamental vibrations have been calculated with imaginary wavenumbers. The reported X-ray crystallographic structure of cyclo(L-Ser-L-Ser), shows that the DKP ring displays a near-planar conformation, with both the two L-Beryl side chains being folded above the ring. It is hypothesized that the crystal packing forces constrain the DKP ring in a planar conformation and it is probable that the lower energy boat conformation may prevail in the aqueous environment. Raman scattering and Fourier-transform infrared (FT-IR) spectra of solid state and aqueous solution samples of cyclo(L-Ser-L-Ser) are reported and discussed. Vibrational band assignments have been made on the basis of comparisons with the calculated vibrational spectra and band wavenumber shifts upon deuteration of labile protons. The experimental Raman and IR results for solid-state samples show characteristic amide I vibrations which are split (Raman:1661 and 1687 cm(-1), IR:1666 and 1680 cm(-1)), possibly due to interactions between molecules in a crystallographic unit cell. The cis amide I band is differentiated by its deuterium shift of ~ 30 cm(-1), which is larger than that previously reported for trans amide I deuterium shifts. A cis amide II mode has been assigned to a Raman band located at 1520 cm(-1). The occurrence of this cis amide II mode at a wavenumber above 1500 cm(-1) concurs with results of previously examined CDAP molecules with low molecular weight substituents on the C-alpha atoms, and is also indicative of a relatively unstrained DKP ring.

Iron organic speciation determination in rainwater using cathodic stripping voltammetry

A sensitive method using Competitive Ligand Exchange-Adsorptive Cathodic Stripping Voltammetry (CLE-ACSV) has been developed to determine for the first time iron (Fe) organic speciation in rainwater over the typical natural range of pH. We have adapted techniques previously developed in other natural waters to rainwater samples, using the competing ligand 1-nitroso-2-naphthol (NN). The blank was equal to 0.17 ± 0.05 nM (n = 14) and the detection limit (DL) for labile Fe was 0.15 nM which is 10–70 times lower than that of previously published methods. The conditional stability constant for NN under rainwater conditions was calibrated over the pH range 5.52–6.20 through competition with ethylenediaminetetraacetic acid (EDTA). The calculated value of the logarithm of β′Fe3+3(NN)β′Fe3+(NN)3 increased linearly with increasing pH according to log β′Fe3+3(NN)=2.4±0.6×pH+11.9±3.5log β′Fe3+(NN)3=2.4±0.6×pH+11.9±3.5 (salinity = 2.9, T = 20 °C). The validation of the method was carried out using desferrioxamine mesylate B (DFOB) as a natural model ligand for Fe. Adequate detection windows were defined to detect this class of ligands in rainwater with 40 μM of NN from pH 5.52 to 6.20. The concentration of Fe-complexing natural ligands was determined for the first time in three unfiltered and one filtered rainwater samples. Organic Fe-complexing ligand concentrations varied from 104.2 ± 4.1 nM equivalent of Fe(III) to 336.2 ± 19.0 nM equivalent of Fe(III) and the logarithm of the conditional stability constants, with respect to Fe3+, varied from 21.1 ± 0.2 to 22.8 ± 0.3. This method will provide important data for improving our understanding of the role of wet deposition in the biogeochemical cycling of iron.

The Unexpected Preference for the fac-Isomer with the Tris(5-ester-substituted-2,2′-bipyridine) Complexes of Ruthenium(II)

The synthesis of a number of new 2,2'-bipyridine ligands, functionalized with bulky ester side groups is reported (L2 - L8). Their reaction with [Ru(DMSO)4Cl2] gives rise to tris-chelate ruthenium(II) metal complexes which show an unusually high proportion of the fac-isomer, as judged by 1H NMR following conversion to the ruthenium(II) complex of 2,2'-bipyridine-5-carboxylic acid methyl ester (L1). The initial reaction appears to have thermodynamic control with the steric bulk of the ligands causing the third ligand to be labile under the reaction conditions used, giving rise to disappointing yields and allowing rearrangement to the more stable facial form. DFT studies indicate that this does not appear to be as a consequence of a metal centered electronic effect. The two isomers of [Ru(L1)3](PF6)2 were separated into the two individual forms using silica preparative plate chromatographic procedures, and the photophysical characteristics of the two forms compared. The results appear to indicate that there is no significant difference in both their room temperature electronic absorption and emission spectra or their excited state lifetimes at 77K.

Synthesis of N3- and 2-NH2-substituted 6,7-diphenylpterins and their use as intermediates for the preparation of oligonucleotide conjugates designed to target photooxidative damage on single-stranded DNA representing the bcr-abl chimeric gene

Two 17-mer oligodeoxynucleotide-5'-linked-(6,7-diphenylpterin) conjugates, 2 and 3, were prepared as photosensitisers for targeting photooxidative damage to a 34-mer DNA oligodeoxynucleotide (ODN) fragment 1 representing the chimeric bcr-abl gene that is implicated in the pathogenesis of chronic myeloid leukaemia (CML). The base sequence in the 17-mer was 3'G G T A G T T A T T C C T T C T T5'. In the first of these ODN conjugates (2) the pterin was attached at its N3 atom, via a -(CH2)3OPO(OH)- linker, to the 5'-OH group of the ODN. Conjugate 2 was prepared from 2-amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone 10, using phosphoramidite methodology. Starting material 10 was prepared from 5-amino-7-methylthiofurazano[3,4-d]pyrimidine 4 via an unusual highly resonance stabilised cation 8, incorporating the rare 2H,6H-pyrimido[6,1-b][1,3]oxazine ring system. In the characterisation of 10 two pteridine phosphazenes, 15 and 29, were obtained, as well as new products containing two uncommon tricyclic ring systems, namely pyrimido[2,1-b]pteridine (20 and 24) and pyrimido[1,2-c]pteridine (27). In the second ODN conjugate the linker was -(CH2)5CONH(CH2)6OPO(OH)- and was attached to the 2-amino group of the pterin. In the preparation of 3, the N-hydroxysuccinimide ester 37 of 2-(5-carboxypentylamino)-6,7-diphenyl-4(3H)-pteridinone was condensed with the hexylamino-modified 17-mer. Excitation of 36 with near UV light in the presence of the single-stranded target 34-mer, 5'T G A C C A T C A A T A A G14 G A A G18 A A G21 C C C T T C A G C G G C C3' 1 caused oxidative damage at guanine bases, leading to alkali-labile sites which were monitored by polyacrylamide gel electrophoresis. Cleavage was observed at all guanine sites with a marked preference for cleavage at G14. In contrast, excitation of ODN-pteridine conjugate 2 in the presence of 1 caused oxidation of the latter predominantly at G18, with a smaller extent of cleavage at G15 and G14 (in the double-stranded portion) and G21. These results contrast with our previous observation of specific cleavage at G21 with ruthenium polypyridyl sensitisers, and suggest that a different mechanism, probably one involving Type 1 photochemical electron transfer, is operative. Much lower yields were found with the ODN-pteridine conjugate 3, perhaps as a consequence of the longer linker between the ODN and the pteridine in this case.