Exploring privileged structures: the combinatorial synthesis of cyclic peptides

Head-to-tail cyclic peptides have been reported to bind to multiple, unrelated classes of receptor with high affinity. They may therefore be considered to be privileged structures. This review outlines the strategies by which both macrocyclic cyclic peptides and cyclic dipeptides or diketopiperazines have been synthesised in combinatorial libraries. It also briefly outlines some of the biological applications of these molecules, thereby justifying their inclusion as privileged structures.

Ephrin-A5 induces rounding, blebbing and deadhesion of EphA3-expressing 293T and melanoma cells by CrkII and Rho-mediated signalling

Eph receptor tyrosine kinases and ephrins regulate morphogenesis in the developing embryo where they effect adhesion and motility of interacting cells. Although scarcely expressed in adult tissues, Eph receptors and ephrins are overexpressed in a range of tumours. In malignant melanoma, increased Eph and ephrin expression levels correlate with metastatic progression. We have examined cellular and biochemical responses of EphA3-expressing melanoma cell lines and human epithelial kidney 293T cells to stimulation with polymeric ephrin-A5 in solution and with surfaces of defined ephrin-A5 densities. Within minutes, rapid reorganisation of the actin and myosin cytoskeleton occurs through activation of RhoA, leading to the retraction of cellular protrusions, membrane blebbing and detachment, but not apoptosis. These responses are inhibited by monomeric ephrin-A5, showing that receptor clustering is required for this EphA3 response. Furthermore, the adapter CrkII, which associates with tyrosine-phosphorylated EphA3 in vitro, is recruited in vivo to ephrin-A5-stimulated EphA3. Expression of an SH3-domain mutated CrkII ablates cell rounding, blebbing and detachment. Our results suggest that recruitment of CrkII and activation of Rho signalling are responsible for EphA3-mediated cell rounding, blebbing and de-adhesion, and that ephrin-A5-mediated receptor clustering and EphA3 tyrosine kinase activity are essential for this response.

Antagonists of protein kinase C inhibit rat retinal glutamate transport activity in situ

Neuronal and glial high-affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by following the fate of the non-metabolisable glutamate transporter substrate, D-aspartate. In the rat retina, pan-isoform PKC inhibition with chelerythrine suppressed glutamate uptake by GLAST (glutamate/aspartate transporter), the dominant excitatory amino acid transporter localized to the glial Muller cells. This effect was mimicked by rottlerin but not by Go6976, suggesting the involvement of the PKCdelta isoform, but not PKCalpha, beta or gamma. Western blotting and immunohistochemical labeling revealed that the suppression of glutamate transport was not due to a change in transporter expression. Inhibition of PKCdelta selectively suppressed GLAST but not neuronal glutamate transporter activity. These data suggest that the targeting of specific glutamate transporters with isoform-specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis, e.g. glaucoma and amyotrophic lateral sclerosis.

Localization of taurine transporters, taurine, and H-3 taurine accumulation in the rat retina, pituitary, and brain

The nervous system contains an abundance of taurine, a neuroactive sulfonic acid. Antibodies were generated against two cloned high-affinity taurine transporters, referred to in this study as TAUT-1 and TAUT-2. The distribution of such was compared with the distribution of taurine in the rat brain, pituitary, and retina. The cellular pattern of [H-3] taurine uptake in brain slices, pituitary slices, and retinas was examined by autoradiography. TAUT-2 was predominantly associated with glial cells, including the Bergmann glial cells of the cerebellum and astrocytes in brain areas such as hippocampus. Low-level labeling for TAUT-2 was also observed in some neurones such as CA1 pyramidal cells. TAUT-1 distribution was more limited; in the posterior pituitary TAUT-1 was associated with the pituicytes but was absent from glial cells in the intermediate and anterior lobes. Conversely, in the brain TAUT-1 was associated with cerebellar Purkinje cells and, in the retina, with photoreceptors and bipolar cells. Our data suggest that intracellular taurine levels in glial cells and neurons may be regulated in part by specific high-affinity taurine transporters. The heterogeneous distribution of taurine and its transporters in the brain does not reconcile well with the possibility that taurine acts solely as a ubiquitous osmolyte in nervous tissues. (C) 2002 Wiley-Liss, Inc.

14-3-3 acts as an intramolecular bridge to regulate cdc25B localization and activity

One of the major regulators of mitosis in somatic cells is cdc25B. cdc25B is tightly regulated at multiple levels. The final activation step involves the regulated binding of 14-3-3 proteins. Previous studies have demonstrated that Ser-323 is a primary 14-3-3 binding site in cdc25B, which influences its activity and cellular localization. 14-3-3 binding to this site appeared to interact with the N-terminal domain of cdc25B to regulate its activity. The presence of consensus 14-3-3 binding sites in the N-terminal domain suggested that the interaction is through direct binding of the 14-3-3 dimer to sites in the N-terminal domain. We have identified Ser-151 and Ser-230 in the N-terminal domain as functional 14-3-3 binding sites utilized by cdc25B in vivo. These low affinity sites cooperate to bind the 14-3-3 dimer bound to the high affinity Ser-323 site, thus forming an intramolecular bridge that constrains cdc25B structure to prevent access of the catalytic site. Loss of 14-3-3 binding to either N-terminal site relaxes cdc25B structure sufficiently to permit access to the catalytic site, and the nuclear export sequence located in the N-terminal domain. Mutation of the Ser-323 site was functionally equivalent to the mutation of all three sites, resulting in the complete loss of 14-3-3 binding, increased access of the catalytic site, and access to nuclear localization sequence.

Identification and characterization of the hepatic stellate cell transferrin receptor

Activated hepatic stellate cells have been implicated in the fibrogenic process associated with iron overload, both in animal models and in human hemochromatosis. Previous studies have evaluated the role of ferritin/ferritin receptor interactions in the activation of stellate cells and subsequent fibrogenesis; however, the role of transferrin in hepatic stellate cell biology is unknown. This study was designed to identify and characterize the stellate cell transferrin receptor and to evaluate the influence of transferrin on stellate cell activation. Identification and characterization of the stellate cell transferrin receptor was determined by competitive displacement assays. The effect of transferrin on stellate cell activation was assessed using western blot analysis for alpha-smooth muscle actin expression, [H-3]Thymidine incorporation, and real-time RT-PCR for procollagen 1(I) mRNA expression. A specific receptor for rat transferrin was observed on activated but not quiescent stellate cells. Transferrin significantly increased the expression of alpha-smooth muscle actin, but caused a decrease in proliferation. Transferrin induced a significant increase in procollagen alpha1(I) mRNA expression. In conclusion, this study has demonstrated for the first time a specific, high affinity receptor for rat transferrin on activated hepatic stellate cells, which via interaction with transferrin regulates stellate cell activation. This suggests that transferrin may be an important factor in the activation of hepatic stellate cells in conditions of iron overload.

Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4

Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1. families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1 L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c-both tagged and detagged-from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies. Crown copyright (C) 2003 Published by Elsevier Inc. All rights reserved.

Caracterização do papel da adrenalina na maturação dos adrenorreceptores B2

As respostas pós-juncionais mediadas por adrenorreceptores β2 (ARβ2), responsáveis pelo relaxamento do músculo liso, na veia safena do cão, estão ausentes à nascença. Pelo contrário, no rato recém-nascido já se verifica a estimulação da adenilil ciclase pela activação dos ARβ2. Não existem ainda estudos no coelho recém-nascido. O principal objectivo deste trabalho é avaliar as respostas pós-juncionais mediadas pelos ARβ2 em coelhos recém-nascidos e jovens e relacionar essas respostas com a adrenalina produzida nas glândulas supra-renais. Traçaram-se curvas de dose-resposta à isoprenalina (agonista β) utilizando-se anéis de aorta montados em banho de órgãos isolados ligado a um transdutor de força isométrica. As catecolaminas das supra-renais foram quantificadas por RP-HPLC-ED. Em aortas pré-contraídas com fenilefrina (agonista α1), a isoprenalina causou relaxamento total apenas em coelhos recém-nascidos (n=10). O relaxamento máximo nos coelhos jovens foi de 21±4% (n=23). A potência da isoprenalina foi maior nos recém-nascidos (EC50=1.15×10-8±7.2×10-10 M, n=10) do que nos coelhos jovens (EC50=1.29×10-7 ±4.7×10-9 M, n=23). O relaxamento máximo com isoprenalina, em aortas pré-contraídas com prostaglandina F2α (PGF2α), no grupo de coelhos recém-nascidos foi de 95±3.6% (n=16). O relaxamento máximo nos coelhos jovens foi de 43.7±8.6% (n=9). Na pré-contracção com PGF2α a potência da isoprenalina registou-se maior nos recémnascidos (EC50=9.59×10-9±4.0×10-10 M, n=16) do que nos coelhos jovens (EC50=2.13×10- 8±3.8×10-9 M, n=9), estando concordante com os resultados da pré-contracção com fenilefrina. Nas supra-renais dos recém-nascidos, o conteúdo de noradrenalina foi de 586±128 nmol/mg e da adrenalina foi de 1915±356 nmol/mg (n=4) e nos coelhos jovens foi de 112±12 nmol/mg e de 3644±403 nmol/mg (n=6), respectivamente. As respostas mediadas por ARβ2 no coelho desenvolvem-se mais cedo do que no cão, pois já estão presentes no nascimento. Tal como no rato, no coelho a adrenalina é já a catecolamina em maior quantidade à nascença, enquanto no cão é vestigial. Há uma relação temporal entre a síntese da adrenalina, a única catecolamina biogénica com alta afinidade para os ARβ2 e a maturação das respostas pós-juncionais mediadas por esses receptores. Um protocolo para experiências futuras destinadas a testar esta hipótese, com base no knockdown da Feniletanolamina-N-metiltransferase por RNAi foi elaborado e incluído neste documento.

Wetting behaviour of femtosecond laser textured Ti-6Al-4V surfaces

The aim of the present work was to investigate the wetting behaviour of biomedical grade Ti-6Al-4V alloy surfaces textured by a femtosecond laser treatment. The material was treated in ambient atmosphere using an Yb: KYW chirped-pulse-regenerative amplification laser with a wavelength of 1030 nm and a pulse duration of 500 fs. Four main types of surface textures were obtained depending on the processing parameters and laser treatment method. These textures consist of: (1) nanoscale laser-induced periodic surface structures (LIPSS); (2) nanopillars; (3) a bimodal roughness distribution texture formed of LIPSS overlapping microcolumns; (4) a complex texture formed of LIPSS overlapping microcolumns with a periodic variation of the columns size in the laser scanning direction. The wettability of the surfaces was evaluated by the sessile drop method using distilled-deionized (DD) water and Hank's balanced salt solution (HBSS) as testing liquids. The laser treated surfaces present a hydrophilic behaviour as well as a high affinity for the saline solution, with equilibrium contact angles in the ranges 24.1-76.2. for DD water and 8.4-61.8. for HBSS. The wetting behaviour is anisotropic, reflecting the anisotropy of the surface textures. (c) 2012 Elsevier B.V. All rights reserved.

The expression in saccharomyces cerevisiae of a glucose/xylose symporter from Candida intermedia is affected by the presence of a glucose/xylose facilitator

Microbiology, 154

Influence of reconstruction parameters for FBP in semiquantification of brain studies with 123I-FPCIT

Brain dopamine transporters imaging by Single Photon Emission Tomography (SPECT) with 123I-FP-CIT has become an important tool in the diagnosis and evaluation of parkinsonian syndromes, since this radiopharmaceutical exhibits high affinity for membrane transporters responsible for cellular reabsorption of dopamine on the striatum. However, Ordered Subset Expectation Maximization (OSEM) is the method recommended in the literature for imaging reconstruction. Filtered Back Projection (FBP) is still used due to its fast processing, even if it presents some disadvantages. The aim of this work is to investigate the influence of reconstruction parameters for FBP in semiquantification of Brain Studies with 123I-FPCIT compared with those obtained with OSEM recommended reconstruction.

New modified electrochemical conductive paper support for BSA detection

Chemical sensors and biosensors are widely used to detect various kinds of protein target biomolecules. Molecularly Imprinted Polymers (MIPs) have raised great interest in this area, because these act as antibody-like recognition materials, with high affinity to the template molecule. Compared to natural antibodies, these are also of lower cost and higher stability. There are different types of supports used to carry MIP materials, mostly of these made of gold, favourably assembled on a Screen Printed Electrode (SPE) strategy. For this work a new kind of support for the sensing layer was developed: conductive paper. This support was made by modifying first cellulose paper with paraffin wax (to make it waterproof), and casting a carbon-ink on it afterwards, to turn it conductive. The SPAM approach previously reported in1 was employed herein to assemble to MIP sensing material on the conductive paper. The selected charged monomers were (vinylbenzyl) trimethlammonium chloride (positive charge) or vinylbenzoic acid (negative charge), used to generate binding positions with single-type charge (positive or negative). The non-specific binding area of the MIP layer was assembled by chronoamperometry-assisted polymerization (at 1 V, for 60, 120 or 180 seconds) of vinylbenzoate, cross-linked with ethylene glycol vinyl ether. The BSA biomolecules lying within the polymeric matrix were removed by Proteinase K action. All preparation stages of the MIP assembly were followed by FTIR, Raman spectroscopy and, electrochemical analysis. In general, the best results were obtained for longer polymerization times and positively charged binding sites (which was consistent with a negatively-charged protein under physiological pH, as BSA). Linear responses against BSA concentration ranged from 0.005 to 100 mg/mL, in PBS buffer standard solutions. The sensor was further calibrated in standard solutions that were prepared in synthetic or real urine, and the analytical response became more sensitive and stable. Compared to the literature, the detection capability of the developed device is better than most of the reported electrodes. Overall, the simplicity, low cost and good analytical performance of the BSA SPE device, prepared with positively charged binding positions, seems a suitable approach for practical application in clinical context. Further studies with real samples are required, as well as gathering with electronic-supporting devices to allow on-site readings.

Neurotoxic activity and ultrastructural changes in muscles caused by the brown widow spider Latrodectus geometricus venom

Brown widow spider (Latrodectus geometricus) venom (BrWSV) produces few local lesions and intense systemic reactions such as cramps, harsh muscle pains, nausea, vomiting and hypertension. Approximately 16 protein bands under reducing conditions and ~ 14 bands under non-reducing conditions on a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis were observed. Neurotoxic clinical manifestations were confirmed in vivo, while proteolytic activity was demonstrated on gelatine film. Severe ultrastructural damages in mice skeletal muscles were observed at 3, 6, 12 and 24 h postinjection with at total of 45 µg of venom protein. Infiltration of eosinophils and ruptures of the cellular membranes were observed in the muscles along with swelling of the nuclear cover and interruption of the collagen periodicity. Altered mitochondrias and autophage vacuoles, nuclear indentation and mitochondria without cristae, slight increment of intermyofibrillar and subsarcolemic spaces and myelinic figures formation were also observed. In the capillary, endothelial membrane unfolding into the lumen was noticed; along with myelinic figures compatible with a toxic myopathy. Swollen sarcotubular systems with lysis of membrane, intense mitochondria autophagia and areas without pinocytic vesicles were observed. Swollen mitochondria surrounded by necrotic areas, myofibrillar disorganization and big vacuolas of the sarcotubular system, degenerated mitochondrium with formation of myelinic figure was seen. Glycogenosomes with small particulate, muscle type glycogen was noticed. Autophagic vacuole (autophagolysosomes) and necrotic areas were also noticed. These damages may be due to interactive effects of the multifactorial action of venom components. However, Latrodectus geometricus venom molecules may also be utilized as neuro therapeutic tools, as they affect neuronal activities with high affinity and selectivity. To our knowledge, the present study is the first ultrastructural report in the literature of muscle injuries and neurological and proteolytic activities caused by BrWSV.

Studies on BolA and ribonuclease R: two important factors in the control of bacterial gene expression

Dissertation presented to obtain the Ph.D degree in Biology