Extension of life span and improvement of vitality of Drosophila melanogaster by long-term supplementation with different molecular weight polysaccharides from Porphyra haitanensis

Polysaccharides isolated from Porphyra (porphyran) have been known to have diverse biological activities, including immunomodulatory and antioxidant activities. The molecular weight-antiaging activity relationship of degraded porphyrans was examined in this study. Natural porphyran was extracted from P. haitanensis, and then was degraded into different molecular weight fractions, P1 molecular weight 49 kDa, P2 molecular weight 30 kDa, P3 molecular weight 8.2 kDa, by free radical. The influence on life span and vitality of porphyrans were carried out on Drosophila melanogaster. We found that all the degraded porphyrans and natural porphyran (P), added daily to the diet, can significantly increase the life span of D. melanogaster, except for P3. Among them, P1 exhibited the most prolonging life span activity. Furthermore, vitality of middle-aged flies (assessed by measuring their mating capacity) receiving porphyrans was increased considerably in comparison with the controls. Finally, in the heat-stress test, we observed a remarkable increase in survival time, especially in P3-diet groups. These results suggest that porphyrans may be effective in reducing the rate of the aging process and molecular weight has important influence on the effects. It seems that P1 and P2, possessed higher molecular weight, may be more useful in normal metabolic condition and P3, possessed the lowest molecular weight, may be more beneficial for D. melanogaster in stress condition. (C) 2007 Elsevier Ltd. All rights reserved.

Crystal structure and DFT studies of a triazole derivative: 4-(2-hydrobenzylideneamino)-3-(1,2,4-triazol-4-yl-methyl) 1H-1,2,4-triazole-5 (4H)-thione

A novel triazole derivative 4-(2-hydrobenzylideneamino)-3-(1, 2, 4-triazol-4-ylmethyl)-1H-1, 2, 4-triazole-5 (4H)-thione(1) was synthesized and characterized using elemental analysis, MR, and H-1 NMR, and its crystal structure was determined via X-ray single crystal diffraction analysis. Crystal data: monoclinic, P2 (1)/c, a = 0.83335 (9) nm, b = 1. 49777 (16) run, c = 1. 14724 (12) nm, beta = 107. 990 (2)degrees, D = 1. 470 Mg/m(3), and Z = 4. The geometries and the vibrational frequencies were determined using the density functional theory(DFT) method at the B3LYP/6-31G* level. To demonstrate the accuracy of the reaction route of compound 1, one of the important intermediates was also tested using the same method. The structural parameters of the two compounds calculated using the DFT study are close to those of the crystals, and the harmonic vibrations of the two compounds computed via the DFT method are in good agreement with those in the observed IR spectral data. The thermodynamic properties of the title compound were calculated, and the compound shows a good structural stability at normal temperature. The test results of biological activities show that it has a certain bactericidal ability.

Inibidores da renina

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

Crystal engineering: exploiting variation of sulfur oxidation for the control of the solid state structure of organosulfur compounds

This thesis is focused on the design and synthesis of a diverse range of novel organosulfur compounds (sulfides, sulfoxides and sulfones), with the objective of studying their solid state properties and thereby developing an understanding of how the molecular structure of the compounds impacts upon their solid state crystalline structure. In particular, robust intermolecular interactions which determine the overall structure were investigated. These synthons were then exploited in the development of a molecular switch. Chapter One provides a brief overview of crystal engineering, the key hydrogen bonding interactions utilized in this work and also a general insight into “molecular machines” reported in the literature of relevance to this work. Chapter Two outlines the design and synthetic strategies for the development of two scaffolds suitable for incorporation of terminal alkynes, organosulfur and ether functionalities, in order to investigate the robustness and predictability of the S=O•••H-C≡C- and S=O•••H-C(α) supramolecular synthons. Crystal structures and a detailed analysis of the hydrogen bond interactions observed in these compounds are included in this chapter. Also the biological activities of four novel tertiary amines are discussed. Chapter Three focuses on the design and synthesis of diphenylacetylene compounds bearing amide and sulfur functionalities, and the exploitation of the N-H•••O=S interactions to develop a “molecular switch”. The crystal structures, hydrogen bonding patterns observed, NMR variable temperature studies and computer modelling studies are discussed in detail. Chapter Four provides the overall conclusions from chapter two and chapter three and also gives an indication of how the results of this work may be developed in the future. Chapter Five contains the full experimental details and spectral characterisation of all novel compounds synthesised in this project, while details of the NCI (National Cancer Institute) biological test results are included in the appendix.

Identifying novel molecular mechanisms of ghrelin receptor signalling underlying neural control of food intake: interaction with stress and impulsivity

The gut-hormone, ghrelin, activates the centrally expressed growth hormone secretagogue 1a (GHS-R1a) receptor, or ghrelin receptor. The ghrelin receptor is a G-protein coupled receptor (GPCR) expressed in several brain regions, including the arcuate nucleus (Arc), lateral hypothalamus (LH), ventral tegmental area (VTA), nucleus accumbens (NAcc) and amygdala. Activation of the GHS-R1a mediates a multitude of biological activities, including release of growth hormone and food intake. The ghrelin signalling system also plays a key role in the hedonic aspects of food intake and activates the dopaminergic mesolimbic circuit involved in reward signalling. Recently, ghrelin has been shown to be involved in mediating a stress response and to mediate stress-induced food reward behaviour via its interaction with the HPA-axis at the level of the anterior pituitary. Here, we focus on the role of the GHS-R1a receptor in reward behaviour, including the motivation to eat, its anxiogenic effects, and its role in impulsive behaviour. We investigate the functional selectivity and pharmacology of GHS-R1a receptor ligands as well as crosstalk of the GHS-R1a receptor with the serotonin 2C (5-HT2C) receptor, which represent another major target in the regulation of eating behaviour, stress-sensitivity and impulse control disorders. We demonstrate, to our knowledge for the first time, the direct impact of GHS-R1a signalling on impulsive responding in a 2-choice serial reaction time task (2CSRTT) and show a role for the 5-HT2C receptor in modulating amphetamine-associated impulsive action. Finally, we investigate differential gene expression patterns in the mesocorticolimbic pathway, specifically in the NAcc and PFC, between innate low- and high-impulsive rats. Together, these findings are poised to have important implications in the development of novel treatment strategies to combat eating disorders, including obesity and binge eating disorders as well as impulse control disorders, including, substance abuse and addiction, attention deficit hyperactivity disorder (ADHD) and mood disorders.

Central nervous system drugs: Low temperature X-ray structures of (I) the base 5-(2,3-dichlorophenyl)-2,4-diamino-6-fluoromethyl-pyrimidine (4030W92) and (II) 5-(2,6-dichlorophenyl)-1-H-2,4-diamino-6-methyl-pyrimidine methanesulphonic acid salt (227C89)

The X-ray crystal structures of (I), the base 4030W92, 5-(2,3-dichlorophenyl)-2,4-diamino-6-fluoromethyl-pyrimidine, C11H9Cl2FN4, and (II) 227C89, the methanesulphonic acid salt of 5-(2,6-dichlorophenyl)-1-H-2,4-diamino-6-methyl-pyrimidine, C11H11Cl2N4 center dot CH3O3S, have been carried out at low temperature. A detailed comparison of the two structures is given. Structure (I) is non-centrosymmetric, crystallizing in space group P2(1) with unit cell a = 10.821(3), b = 8.290(3), c = 13.819(4) angstrom, beta = 105.980(6)degrees, V = 1191.8(6) angstrom(3), Z = 4 (two molecules per asymmetric unit) and density (calculated) = 1.600 mg/m(3). Structure (II) crystallizes in the triclinic space group P (1) over bar with unit cell a = 7.686(2), b = 8.233(2), c = 12.234(2) angstrom, alpha = 78.379(4), beta = 87.195(4), gamma = 86.811(4)degrees, V = 756.6(2) angstrom(3), Z = 2, density (calculated) = 1.603 mg/m(3). Final R indices [I > 2sigma(I)] are R1 = 0.0572, wR2 = 0.1003 for (I) and R1 = 0.0558, wR2 = 0.0982 for (II). R indices (all data) are R1 = 0.0983, wR2 = 0.1116 for (I) and R1 = 0.1009, wR2 = 0.1117 for (II). 5- Phenyl-2,4 diaminopyrimidine and 6-phenyl-1,2,4 triazine derivatives, which include lamotrigine (3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine), have been investigated for some time for their effects on the central nervous system. The three dimensional structures reported here form part of a newly developed data base for the detailed investigation of members of this structural series and their biological activities.

Prooxidant action of knipholone anthrone: Copper dependent reactive oxygen species generation and DNA damage

Knipholone (KP) and knipholone anthrone (KA) are natural 4-phenylanthraquinone structural analogues with established differential biological activities including in vitro antioxidant and cytotoxic properties. By using DNA damage as an experimental model, the comparative Cu(II)-dependent prooxidant action of these two compounds were studied. In the presence of Cu(II) ions, the antioxidant KA (3.1-200 [mu]M) but not KP (6-384 [mu]M) caused a concentration-dependent pBR322 plasmid DNA strand scission. The DNA damage induced by KA could be abolished by reactive oxygen species scavengers, glutathione and catalase as well as EDTA and a specific Cu(I) chelator bathocuproine disulfonic acid. In addition to Cu(II) chelating activity, KA readily reduces Cu(II) to Cu(I). Copper-dependent generation of reactive oxygen species and the subsequent macromolecular damage may be involved in the antimicrobial and cytotoxic activity of KA.

Low temperature X-ray structures of two crystalline forms (I) and (II) of the pyrimidine derivative and sodium channel blocker BW1003C87: 5-(2,3,5-trichlorophenyl)-2,4-diaminopyrimidine

The X-ray crystal structures of two crystalline forms of 5-(2,3,5-trichlorophenyl)-2,4-diaminopyrimidine, C10H7Cl3N4 (code name BW1003C87) (I) and (II), have been carried out at liquid nitrogen temperature. A detailed comparison of the two structures is given. Both are centrosymmetric, with structure (I) in the triclinic space group P (1) over bar unit cell a = 6.4870(10), b = 9.216(2), c = 12.016(2) angstrom, alpha = 75.78(3)degrees, beta = 89.95(3)degrees, gamma = 83.45(3)degrees, V = 691.5(2) angstrom(3), Z = 2 and density (calculated) = 1.544 Mg/m(3); and (II) in the monoclinic space group P2(1)/c, unit cell a = 12.000(2), b = 7.518(2), c = 13.450(3) angstrom, beta = 97.87(3)degrees, V = 1202.0(5) angstrom(3), Z = 4, Density (calculated) = 1.600 Mg/m(3). Structure (I) includes a solvated CH3OH in the lattice. Final R indices [I > 2sigma(I)] are R1 = 0.0427, wR2 = 0.1075 for (I) and R1 = 0.0487, wR2 = 0.1222 for (II). R indices (all data) are R1 = 0.0470, wR2 = 0.1118 for (I) and R1 = 0.0623, wR2 = 0.1299 for (II). 5-Phenyl-2,4 diaminopyrimidine and 6-phenyl-1,2,4 triazine derivatives, which include lamotrigine (3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine), have been investigated for some time for their effects on the central nervous system. Both lamotrigine and 5-(2,3,5-trichlorophenyl)-2,4-diaminopyrimidine (code name BW1003C87), the subject of the present study, are anticonvulsant as well as neuroprotective in models of brain ischaemia and in a model of white matter ischaemia. BW1003C87 is a sodium channel blocker which also reduces the release of the neurotransmitter glutamate. The three dimensional structures reported here form part of a newly developed data base for the detailed investigation of members of this drug family and their biological activities.

Antioxidant activity of Knipholone anthrone

Knipholone (KP) and knipholone anthrone (KA) are natural 4-phenylanthraquinone structural analogues with established differential biological activities including in vitro antioxidant and cytotoxic properties. By using DNA damage as an experimental model, the comparative Cu(II)-dependent prooxidant action of these two compounds were studied. In the presence of Cu(II) ions, the antioxidant KA (3.1-200 microM) but not KP (6-384 microM) caused a concentration-dependent pBR322 plasmid DNA strand scission. The DNA damage induced by KA could be abolished by reactive oxygen species scavengers, glutathione and catalase as well as EDTA and a specific Cu(I) chelator bathocuproine disulfonic acid. In addition to Cu(II) chelating activity, KA readily reduces Cu(II) to Cu(I). Copper-dependent generation of reactive oxygen species and the subsequent macromolecular damage may be involved in the antimicrobial and cytotoxic activity of KA.

Interactions of the 67 kDa laminin receptor and its precursor with laminin

The 67LR (67 kDa laminin receptor) enables cells to interact with components of the extracellular matrix The molecule is derived from the 37LRP (37 kDa laminin receptor precursor); however, the precise molecular mechanism of this conversion is unknown. Recombinant 37LRP expressed in and purified from Escherichia colt, bound to human laminin in a SPR (surface plasmon resonance) experiment. 67LR isolated from human breast-cancer-derived cells in culture was also shown to bind to laminin by SPR. However, the kinetics of association are qualitatively different. 37LRP but not 67LR, binds to heparan sulfate. The binding of 37LRP to heparan sulfate did not affect the interaction of 37LRP with laminin In contrast, heparan sulfate reduces the extent of binding of laminin to 67LR. Taken together, these results show that 37LRP has some of the biological activities of 67LR, even prior to the conversion event. However, the conversion affects the sites of interaction with both laminin and heparan sulfate.

Cytosol protein regulation in H295R steroidogenesis model induced by the zearalenone metabolites, α- and β-zearalenol.

a- and b-zearalenol (a-ZOL and b-ZOL, respectively) are metabolites of the mycotoxin zearalenone (ZEN). All three individual mycotoxins have shown to be biological active i.e. being estrogenic and able to stimulate cellular proliferation albeit at different strengths. In this work, cytosol protein expression was determined by using stable-isotope labelling by amino acids in cell culture (SILAC) upon exposure of a-ZOL and b-ZOL to the steroidogenesis cell model H295R. A total of 14 and 5 individual proteins were found to be significantly regulated by a-ZOL and b-ZOL, respectively. Interestingly, there were no common protein regulations by the metabolites or the parent mycotoxin ZEN. Furthermore, the regulated proteins were assigned to networks and groups of actions that also differed from one another suggesting that the three individual mycotoxins may have unique biological activities.

Burkholderia cenocepacia BC2L-C Is a Super Lectin with Dual Specificity and Proinflammatory Activity

Lectins and adhesins are involved in bacterial adhesion to host tissues and mucus during early steps of infection. We report the characterization of BC2L-C, a soluble lectin from the opportunistic pathogen Burkholderia cenocepacia, which has two distinct domains with unique specificities and biological activities. The N-terminal domain is a novel TNF-alpha-like fucose-binding lectin, while the C-terminal part is similar to a superfamily of calcium-dependent bacterial lectins. The C-terminal domain displays specificity for mannose and L-glycero-D-manno-heptose. BC2L-C is therefore a superlectin that binds independently to mannose/heptose glycoconjugates and fucosylated human histo-blood group epitopes. The apo form of the C-terminal domain crystallized as a dimer, and calcium and mannose could be docked in the binding site. The whole lectin is hexameric and the overall structure, determined by electron microscopy and small angle X-ray scattering, reveals a flexible arrangement of three mannose/heptose-specific dimers flanked by two fucose-specific TNF-alpha-like trimers. We propose that BC2L-C binds to the bacterial surface in a mannose/heptose-dependent manner via the C-terminal domain. The TNF-alpha-like domain triggers IL-8 production in cultured airway epithelial cells in a carbohydrate-independent manner, and is therefore proposed to play a role in the dysregulated proinflammatory response observed in B. cenocepacia lung infections. The unique architecture of this newly recognized superlectin correlates with multiple functions including bacterial cell cross-linking, adhesion to human epithelia, and stimulation of inflammation.

Selectivity fields: comparative molecular field analysis (CoMFA) of the glycine/NMDA and AMPA receptors

An approach for evaluation of binding selectivity was suggested and exemplified using glycine/NMDA and AMPA receptors. For analyzing the pairwise selectivity, we propose to use the difference between biological activities (expressed as -log Ki) of ligands with respect to different receptor subtypes as a dependent variable for building comparative molecular field analysis (CoMFA) models. The resulting fields (which will be referred to as the "selectivity fields") indicate the ways of increasing selectivity of binding, inhibition, etc. As an example, CoMFA of a set of pyrazolo[1,5-c]quinazolines and triazolo[1,5-c]quinazolines was used for considering the binding selectivity with respect to glycine/NMDA and AMPA receptors. In addition, the mapping of these fields onto the molecular models of the corresponding receptors makes it possible to reveal the reasons for experimentally observed selectivity as well as to suggest additional ways of increasing selectivity.

Cloning and characterisation of three novel disintegrin precursors from the venoms of three Atheris species: Atheris chlorechis, Atheris nitschei and Atheris squamigera

Snake venom constitutes one of the most complex mixtures of naturally-occurring toxic proteins/polypeptides and a large number of these possess very profound biological activities. Disintegrins, that are commonly found in viper venoms, are low molecular weight proteins that usually contain an -Arg-Gly-Asp- (-RGD-) motif that is known to be involved in cell adhesion ligand recognition, binding specifically to cell surface integrin receptors and also exhibiting platelet anti-aggregation activity.

Here, we report for the first time, the successful cloning of three cDNAs encoding disintegrin precursors from lyophilised venom-derived libraries of Atheris chlorechis, Atheris nitschei and Atheris squamigera, respectively. All of these disintegrins belong to the short-coding class and all exhibit high degrees of structural identity, both in their amino acid sequences and in the arrangement of their functional domains. Mass spectrometric analyses of the HPLC-separated/in-gel digested venom proteins was performed to characterise the mature disintegrins as expressed in the venom proteome. Studies on both the structures and conserved sites within these disintegrins are of considerable theoretical interest in the field of biological evolution and in the development of new research tools or novel templates for drug design.

Involvement of mitochondrial and B-RAF/ERK signaling pathways in berberine-induced apoptosis in human melanoma cells

The natural isoquinoline alkaloid berberine exhibits a wide spectrum of biological activities including antitumor activity, but its mechanism of action remains to be fully elucidated. Here, we report that berberine induced apoptosis in human melanoma cells, through a process that involved mitochondria and caspase activation. Berberine-induced activation of a number of caspases, including caspases 3, 4, 7, 8, and 9. Pan-caspase inhibitor, z-VAD-fmk, and caspase-8 and caspase-9 inhibitors prevented apoptosis. Berberine also led to the generation of the p20 cleavage fragment of BAP31, involved in directing proapoptotic signals between the endoplasmic reticulum and the mitochondria. Treatment of SK-MEL-2 melanoma cells with berberine induced disruption of the mitochondrial transmembrane potential, release of cytochrome c and apoptosis-inducing factor from the mitochondria to the cytosol, generation of reactive oxygen species (ROS), and a decreased ATP/ADP ratio. Overexpression of bcl-xL by gene transfer prevented berberine-induced cell death, mitochondrial transmembrane potential loss, and cytochrome c and apoptosis-inducing factor release, but not ROS generation. N-acetyl-L-cysteine inhibited the production of ROS, but did not abrogate the berberine-induced apoptosis. Inhibition of extracellular signal-regulated kinase (ERK) phosphorylation, by using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and reduction of B-RAF levels by silencing RNA induced cell death of SK-MEL-2 cells, and diminished the berberine concentration required to promote apoptosis. These data show that berberine-induced apoptosis in melanoma cells involves mitochondria and caspase activation, but ROS generation was not essential. Our results indicate that inhibition of B-RAF/ERK survival signaling facilitates the cell death response triggered by berberine. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.