Structural and functional characterization of self-assembling amphiphilic oligopeptides

The aim of this thesis was to design amphiphilic peptides that self-assemble into supramolecular structures, like nano-sized vesicles that can be of use for drug delivery purposes. The oligopeptides were produced by recombinant techniques and are shown to spontaneously self-assemble upon hydration into vesicles in which hydrophillic molecules could be entrapped. Detailed analysis of the obtained self-assembled, vesicular structures has provided insight into the peptide secondary conformation and the stabilizing intermolecular interactions, elucidating the self-assembling characteristics. The peptide vesicles were successfully used for the intracellular delivery of drug molecules. This thesis sheds light onto the design and self-assembling behavior of amphiphilic oligopeptide vesicles that can be of use for drug delivery purposes

Ze krijgt toch Somplex? Onderzoek naar de effectiviteit van onderwijsaanpassingen voor hoogbegaafde leerlingen

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Peptide nanocarriers for intracellular delivery of photosensitizers

Previously we have shown that recombinantly produced amphiphilic oligopeptides with amino acid sequence Ac-Ala-Ala-Val-Val-Leu-Leu-Leu-Trp-Glu-Glu spontaneously assemble into nano-sized vesicles with an average diameter of 120 nm. Moreover, peptide vesicles could be stabilized by introducing multiple cysteine residues within the hydrophobic domain of these amphiphilic oligopeptides, allowing the formation of intermolecular disulfide bridges. In this study, the cellular association and internalization of peptide vesicles were assessed. Flow cytometry and confocal laser-scanning microscopy showed that peptide vesicles were internalized by cells predominantly via adsorptive macropinocytosis. Furthermore, the potential of these peptide vesicles as delivery system for photosensitizers was explored. Water-insoluble phthalocyanines could be quantitatively entrapped within the hydrophobic domains of these peptide vesicles. Confocal laser-scanning microscopy analysis showed that internalized peptides co-localized with the phthalocyanine, suggesting that peptide vesicles are internalized in their intact form. Upon illumination, the phthalocyaninecontaining peptide vesicles showed an active photodynamic response towards the cells leading to effective cell killing. In contrast, the free phthalocyanine or empty peptide vesicles did not show any cytotoxicity. In conclusion, this is the first demonstration that peptide vesicles show promise as delivery systems for photosensitizers to be used in photodynamic therapy

Role of the human erythrocyte in generation and storage of asymmetric dimethylarginine

Davids M, van Hell AJ, Visser M, Nijveldt RJ, van Leeuwen PAM, Teerlink T. Role of the human erythrocyte in generation and storage of asymmetric dimethylarginine. Am J Physiol Heart Circ Physiol 302: H1762-H1770, 2012. First published February 24, 2012; doi: 10.1152/ajpheart.01205.2011.-Proteolytic activity in whole blood may lead to release of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). We investigated the role of the human erythrocyte in storage and generation of ADMA in healthy controls (n = 36) and critically ill patients (n = 38). Both free and total (sum of free and protein-incorporated) ADMA were measured. Upon incubation of intact erythrocytes with extracellular ADMA (0 to 40 mu mol/l), equilibrium between intra-and extracellular ADMA was reached within 3 h. Compared with controls, patients had significantly higher basal concentrations of ADMA in plasma (0.88 +/- 0.75 vs. 0.41 +/- 0.07 mu mol/l) and erythrocytes (1.28 +/- 0.55 vs. 0.57 +/- 0.14 mu mol/l). Intracellular and plasma ADMA were significantly correlated in the patient group only (r = 0.834). Upon lysis, followed by incubation at 37 C for 2 h, free ADMA increased sevenfold (to 8.60 +/- 3.61 mu mol/l in patients and 3.90 +/- 0.78 mu mol/l in controls). In lysates of controls, free ADMA increased further to 9.85 +/- 1.35 mu mol/l after 18 h. Total ADMA was 15.43 +/- 2.44 mu mol/l and did not change during incubation. The increase of free ADMA during incubation corresponded to substantial release of ADMA from the erythrocytic protein-incorporated pool (21.9 +/- 4.6% at 2 h and 60.8 +/- 7.6% at 18 h). ADMA was released from proteins other than hemoglobin, which only occurred after complete lysis and was blocked by combined inhibition of proteasomal and protease activity. Neither intact nor lysed erythrocytes mediated degradation of free ADMA. We conclude that intact erythrocytes play an important role in storage of ADMA, whereas upon erythrocyte lysis large amounts of free ADMA are generated by proteolysis of methylated proteins, which may affect plasma levels in hemolysis-associated disease

Conformation and intermolecular interactions of SA2 peptides self-assembled into vesicles.

Previously we have shown that the recombinantly produced SA2 amphiphilic oligopeptide (Ac-Ala-Ala-Val-Val-Leu-Leu-Leu-Trp-Glu-Glu-COOH) self-assembles into nanovesicles (van Hell et al. 2007). In this study, the intermolecular interactions that contribute to the formation of such peptide vesicles are examined. First, analysis of a 3-hydroxyflavone fluorescent probe inserted into the peptide assemblies demonstrated that the peptide self-assembly is based on hydrophobic clustering. The polarity of this hydrophobic microenvironment was comparable to that of negatively charged lipid bilayers. A substantial level of hydration at the hydrophilic-hydrophobic interface was detected, as was further confirmed by tryptophan fluorescence analysis. However, organic solvents such as acetonitrile, tetrahydrofuran, or ethanol could not disrupt SA2 oligopeptide vesicles, whereas these solvents fully disintegrated lipid vesicles. Instead, the SA2 assembly immediately disintegrated in hydrogen breaking solvents such dimethylsulfoxide and dimethylformamide, suggesting the involvement of additional intermolecular interactions via hydrogen bonding. Circular dichroism and Fourier transform infrared spectroscopy excluded well-defined patterns of intramolecular hydrogen bonding and indicated the polyproline type II as the dominant SA2 peptide conformation, which enables intermolecular hydrogen bonding. All-atom computational simulations were used to confirm the presence of such intermolecular hydrogen bonds and degrees of hydration. On the basis of the experimental and computational data presented, we propose a model of an interdigitated peptide assembly that involves intermolecular hydrogen bonding in addition to hydrophobic interactions that stabilize SA2 oligopeptide vesicles

Adições à flora de briófitas de Mato Grosso do Sul, Brasil Additions to the flora of bryophytes from Mato Grosso do Sul State, Brazil

Nesse levantamento foram encontrados 133 táxons no Estado de Mato Grosso do Sul, sendo um pertencente à divisão Anthocerotophyta em um gênero e uma família, 83 pertencentes à divisão Bryophyta, distribuídos em 54 gêneros e 27 famílias, e 49 à divisão Hepatophyta, distribuídos em 27 gêneros e 13 famílias. Destes, 100 (1 antócero, 59 musgos e 40 hepáticas) são novas citações para o Estado.<br>In this taxonomic survey 133 taxa were found in Mato Grosso do Sul State, one belonging to Anthocerotophyta, distributed into one genus and one family, 83 belonging to Bryophyta, distributed into 54 genera and 27 families, and 49 belonging to Hepatophyta, distributed into 27 genera and 13 families. Of these, 100 (one hornworts, 59 mosses and 40 hepatics) are new records for the State

Molecular Biology, Biochemistry and Cellular Physiology of Cysteine Metabolism in Arabidopsis thaliana

Cysteine is one of the most versatile molecules in biology, taking over such different functions as catalysis, structure, regulation and electron transport during evolution. Research on Arabidopsis has contributed decisively to the understanding of cysteine synthesis and its role in the assimilatory pathways of S, N and C in plants. The multimeric cysteine synthase complex is present in the cytosol, plastids and mitochondria and forms the centre of a unique metabolic sensing and signaling system. Its association is reversible, rendering the first enzyme of cysteine synthesis active and the second one inactive, and vice-versa. Complex formation is triggered by the reaction intermediates of cysteine synthesis in response to supply and demand and gives rise to regulation of genes of sulfur metabolism to adjust cellular sulfur homeostasis. Combinations of biochemistry, forward and reverse genetics, structural- and cell-biology approaches using Arabidopsis have revealed new enzyme functions and the unique pattern of spatial distribution of cysteine metabolism in plant cells. These findings place the synthesis of cysteine in the centre of the network of primary metabolism