Dimitra Benaki

The multiple T-maze in vivo testing of the neuroprotective effect of humanin analogues.

Humanin (HN) and its analogues have been shown to protect cells against death induced by various Alzheimers disease (AD) genes and amyloid-beta-peptides in vitro; the analogues [Gly(14)]-HN and colivelin have also been shown to be potent in reversing learning and memory impairment induced by scopolamine or quinuclidinyl benzilate (QNB) in mice or rats in vivo using the Y-maze or multiple T-maze tests. This paper describes the activity of new peptides of the HN family, after i.p. administration, on QNB-induced impairment of spatial memory in the multiple T-maze test in rats. The following peptides have been studied: HN analogues truncated either on the C- or N-terminus, or analogues having a tert-Leu in place of Leu in the central part of the molecule, the active HN core PAGASRLLLLTGEIDLP (RG-PAGA) and its analogues having three or five leucines instead of four, and finally the recently described hybrid peptide colivelin (i.e. a peptide having the activity-dependent neurotrophic factor SALLRSIPA attached to the N-terminus of the active RG-PAGA) and its des-Leu- and plus-Leu-analogues. While the truncated analogues and most of the tert-Leu containing analogues were devoid of activity, the analogues of the RG-PAGA were active, i.e. they reversed the impairment of spatial memory irrespective of the number of Leu present in their sequence. The highest activity was shown by colivelin and its des-Leu-analogue. These results demonstrate the potential of HN analogues in the modulation of the cholinergic system, which plays an important role in the cognitive deficits associated with AD and other neurodegenerative diseases.

The natural olive constituent oleuropein induces nutritional cardioprotection in normal and cholesterol-fed rabbits: comparison with preconditioning.

Ischemic preconditioning, which is mediated by cell signaling molecules, protects the heart from ischemia-reperfusion injury by limiting the infarct size. Oleuropein, the main polyphenolic constituent of olives, reduced the infarct size in normal and cholesterol-fed rabbits when it was administered at a nutritional dose. The aim of the present study was to compare the effects of oleuropein and preconditioning in terms of the cell signaling and metabolism pathways underlying myocardial protection. Rabbits were randomly divided into six groups: the control group received 5 % dextrose for six weeks, the preconditioning group was subjected to two cycles of preconditioning with 5 min ischemia/10 min reperfusion, the O6 group was treated with oleuropein for six weeks, the Chol group was fed a cholesterol-enriched diet and 5 % dextrose for six weeks, and the CholO6 and CholO3 groups were treated with cholesterol and oleuropein for six and three weeks, respectively; oleuropein was dissolved in 5 % dextrose solution and was administered orally at a dose of 20 mg × kg(-1) × day(-1). All animals were subsequently subjected to 30 min myocardial ischemia followed by 10 min of reperfusion. At that time, myocardial biopsies were taken from the ischemic areas for the assessment of oxidative and nitrosative stress biomarkers (malondialdehyde and nitrotyrosine), and determination of phosphorylation of signaling molecules involved in the mechanism of preconditioning (PI3K, Akt, eNOS, AMPK, STAT3). The tissue extracts NMR metabolic profile was recorded and further analyzed by multivariate statistics. Oxidative biomarkers were significantly reduced in the O6, CholO6, and CholO3 groups compared to the control, preconditioning, and Chol groups. Considering the underlying signaling cascade, the phosphorylation of PI3K, Akt, eNOS, AMPK, and STAT-3 was significantly higher in the preconditioning and all oleuropein-treated groups compared to the control and Chol groups. The NMR-based metabonomic study, performed through the analysis of spectroscopic data, depicted differences in the metabolome of the various groups with significant alterations in purine metabolism. In conclusion, the addition of oleuropein to a normal or hypercholesterolemic diet results in a preconditioning-like intracellular effect, eliminating the deleterious consequences of ischemia and hypercholesterolemia, followed by a decrease of oxidative stress biomarkers. This effect is exerted through inducing preconditioning-involved signaling transduction. Nutritional preconditioning may support the low cardiovascular morbidity and mortality associated with the consumption of olive products.

Nanobiotechnology for the Prevention of Dialysis-related Amyloidosis

Abstract:  Dialysis‐related amyloidosis is related to the inefficient removal of β2‐microglobulin (β2‐m) that is mainly responsible for the formation of amyloid fibrils deposited on the joints and in the heart, blood vessels and digestive system. Magnetically assisted hemodialysis (MAHD) can be used for the prevention of dialysis‐related amyloidosis. MAHD is based on ferromagnetic nanoparticle‐targeted binding substance conjugates (FN‐TBS Cs) that should be administered to the patient before the dialysis session. The TBS should have a high affinity for β2‐m so that the conjugates bind with the β2‐m in the bloodstream. The complex FN‐TBS–β2‐m will be selectively removed during dialysis by means of a “magnetic dialyzer” that is installed at the dialysis machine in series to the conventional dialyzer. We have examined the in vitro applicability of MAHD by employing biocompatible Fe3O4 and bovine serum albumin (BSA) as constituents of the FN‐TBS Cs. We evaluated the binding capacity of both bare Fe3O4 FNs and Fe3O4‐BSA Cs for β2‐m concentrations ranging from mild to severe conditions. Finally, we conducted mock‐dialysis experiments for the evaluation of several technical issues related to MAHD. β2‐m is adsorbed onto the Fe3O4‐BSA Cs not only almost instantly, but also very efficiently. The employed Cs do not chemically interact with the materials used in standard dialyzers, as agglomerates were not observed in the capillaries of the conventional dialyzers. MAHD may become an efficient modality for the prevention of dialysis‐related amyloidosis because β2‐m concentrations ranging from mild to severe conditions can be adequately handled.

Detection of interactions of the β-amyloid peptide with small molecules employing transferred NOEs

The interaction of pineal hormone melatonin, the histological dye thioflavin T, and the olive tree polyphenol oleuropein, with the 28 amino acid residue N‐terminal fragment of the β‐amyloid peptide (β‐AP) of Alzheimers disease, [β‐AP(1‐28)], was detected in solution through the observation of transferred NOEs (trNOEs) in 1D and 2D NOE spectroscopy (NOESY) experiments. The trNOE method is applied for the first time in the detection of interactions of soluble β‐AP(1‐28) with small molecules and may provide a means of screening for the identification of possible inhibitors of the formation of neurotoxic β‐AP assemblies. Copyright

Urine metabolomics in neonates with late-onset sepsis in a case-control study

Although late-onset sepsis (LOS) is a major cause of neonatal morbidity and mortality, biomarkers evaluated in LOS lack high diagnostic accuracy. In this prospective, case-control, pilot study, we aimed to determine the metabolic profile of neonates with LOS. Urine samples were collected at the day of initial LOS evaluation, the 3rd and 10th day, thereafter, from 16 septic neonates (9 confirmed and 7 possible LOS cases) and 16 non-septic ones (controls) at respective time points. Urine metabolic profiles were assessed using non-targeted nuclear magnetic resonance spectroscopy and targeted liquid chromatography-tandem mass spectrometry analysis. Multivariate statistical models with data from either analytical approach showed clear separation between the metabolic profiles of septic neonates (both possible and confirmed) and the controls. Metabolic changes appeared to be related to disease progression. Overall, neonates with confirmed or possible LOS exhibited comparable metabolic profiles indicating similar metabolic alternations upon the onset of clinical manifestations. This methodology therefore enabled the discrimination of neonates with LOS from non-septic individuals, providing potential for further research toward the discovery of LOS-related biomarkers.

Conformational analysis of the nonapeptide leuprorelin using NMR and molecular modeling

The nonapeptide Leuprorelin, one of the LHRH agonists, was studied by means of 2D nuclear magnetic resonance spectroscopy and molecular modeling. NOESY spectra in aqueous/deuterated methanol solution (50% H2O/CD3OD) at low temperature (268 K) revealed short-range nOe connectivities (i, i+1), characteristic of flexibility of the molecule. The H N -H N sequential connectivities observed provide evidence that the sequence has the propensity to form a bend involving residues 5 and 6 and the N-terminal segment. The α-proton chemical shifts compared to random coil and additional data from the amide proton temperature coefficients support this assumption. One long-range nOe cross peak between H 2 α -H NEth is indicative of proximity between C- and N-termini.

Design and synthesis of purine analogues as highly specific ligands for FcyB, a ubiquitous fungal nucleobase transporter

In the course of our study on fungal purine transporters, a number of new 3-deazapurine analogues have been rationally designed, based on the interaction of purine substrates with the Aspergillus nidulans FcyB carrier, and synthesized following an effective synthetic procedure. Certain derivatives have been found to specifically inhibit FcyB-mediated [3H]-adenine uptake. Molecular simulations have been performed, suggesting that all active compounds interact with FcyB through the formation of hydrogen bonds with Asn163, while the insertion of hydrophobic fragments at position 9 and N6 of 3-deazaadenine enhanced the inhibition.

Complexes of an Alpha Thymosin Derivative with 185/187Re and 99mTc: Structural Analysis and Initial Biological Evaluation

Alpha thymosins are a family of immunostimulating peptides first isolated from thymus gland. In the present work, the structure of the ReO(V)3+ complex of an alpha 1 thymosin derivative containing the metal‐chelating N,N‐dimethylglycyl‐l‐seryl‐l‐cysteinyl group was studied with NMR, CD, and ESI. The analysis indicated the existence of two interconverting diastereomers depending on the orientation of the side chain of the chelated Ser syn‐ or anti‐ to the oxygen of the ReO(V)3+ core. The two diastereomers could be separated on HPLC under a slow gradient showing the ratio of syn/anti to be 3:2, in agreement with the NMR data. The conversion process was shown to involve the coordination of a water molecule to the ReO(V)3+ core through the incubation of the complex in 18O‐enriched water and subsequent ESI analysis. HPLC analysis of the analogous radioactive 99mTcO(V)3+ complex showed the formation of two isomers in the same syn/anti 3:2 ratio. Biodistribution studies of the 99mTcO(V)3+ complex in Swiss albino mice with experimentally induced inflammation showed higher accumulated radioactivity in inflamed tissue compared to normal (ratio of inflamed/control tissue 3.9). 99mTc‐labeled complexes of alpha thymosin derivatives are expected to facilitate research on alpha thymosins and accelerate exploitation of these peptides in immunotherapy protocols.

Surveying the response of transport channels of intact RBC membranes upon AgNO3 administration: an atomic force microscopy study.

Background/Aims: Cell membranes facilitate the transport of water, ions, and necessary nutrients by hosting a great variety of transport channels that have either a ‘simple’ pore-like structure or more complex architecture that is based on the utilization of specific receptors. The present study reveals the impact of AgNO3, a well-known inhibitor of water channel activity, on transport channels that emerge at the membrane of intact red blood cells (iRBCs). Methods: Atomic force microscopy is employed to survey the morphological modification of all transport channels by directly comparing the respective images obtained on the exact same iRBCs prior to and after spraying the AgNO3 solution. Results: Small pores of mean size 50 nm that were assigned to water channels, and extended orifices of mean size 300 nm that exhibit a neck-like extracellular segment were observed at the iRBC membrane. Conclusion: Our results reveal that AgNO3 exerts noticeable influence on all transport channels so that its selective water channel inhibitory action should be reconsidered. For low AgNO3 concentrations extended recovery of the small pore network was observed upon waiting, giving strong evidence that iRBCs have a recovery potential upon simply removing the inhibition cause without the need for specific reducing agents.

Structure–activity relationships of αIIb 313–320 derived peptide inhibitors of human platelet aggregation

The αIIbβ3 receptor, which is the most abundant receptor on the surface of platelets, can interact with a variety of adhesive proteins including fibrinogen, fibronectin and the von Willebrand factor. Fibrinogen binding on αIIbβ3 is an event essential for platelet aggregation and thrombus formation. Mapping of the fibrinogen‐binding domains on αIIb subunit suggested the sequence 313–332 as a possible binding site. This region was restricted to sequence αIIb 313–320 (Y313MESRADR320) using synthetic octapeptides overlapping by six residues. The YMESRADR octapeptide inhibits ADP‐stimulated human platelets aggregation and binds to immobilized fibrinogen. In this study, we used the Ala scanning methodology within the sequence 313–320 aiming to evaluate the contribution of each amino acid in inhibiting platelet aggregation. It was found that the substitution of Y313, M314, E315 or S316 by A does not affect the activity of the parent octapeptide. The–RADR‐motif seems to be the most essential for the biological activity of the αIIb 313–320 site. The conformational analysis of the YAESRADR, YMESAADR and YMESRAAR analogs by using NMR spectroscopy and distance geometry calculations revealed significant differences in their conformational states in DMSO‐d6. Copyright