Paul Cordopatis

Effect of a polyphenol-rich wild blueberry extract on cognitive performance of mice, brain antioxidant markers and acetylcholinesterase activity

The aim of this study was to examine the effect of a polyphenol-rich extract (PrB) of Vaccinium angustifolium (wild blueberries) introduced intraperitoneally (i.p.) at 30 (PrB30) and 60 (PrB60) mg/kg body weight for 7 days, on cognitive performance, brain oxidative status and acetylcholinesterase activity in adult, male, 3-4-month-old Balb-c mice. Evaluation of rodent learning and memory was assessed by a step-through test on day 6 after a double training and an initial acquisition trial on day 5. Antioxidant status was determined by ferric reducing antioxidant power (FRAP), ascorbic acid concentration (FRASC), malondialdehyde and reduced glutathione levels in whole brain homogenates. Acetylcholinesterase (AChE) activity was determined by Ellmans colorimetric method. Results showed that the PrB60-treated mice exhibited a significant improvement in learning and memory (step-through latency time of 228+/-38 s compared to 101+/-32 s of the control group). PrB extract administration also resulted in reduced lipid peroxidation products (38 and 79%) and higher brain ascorbic acid levels (21 and 64%) in both PrB30 and PrB60-treated groups, respectively, and higher glutathione levels (28%) in the PrB60-treated group. Furthermore, salt- and detergent soluble AChE activity significantly decreased in both PrB-treated groups. Thus, the significant cognitive enhancement observed in adult mice after short-term i.p. supplementation with the blueberry extract concentrated in polyphenols, is closely related to higher brain antioxidant properties and inhibition of AChE activity. These findings stress the critical impact of wild blueberry bioactive components on brain function.

Memory enhancing effects of saffron in aged mice are correlated with antioxidant protection.

Brain aging is characterized by cognitive decline and memory deficits that could be the result of oxidative stress and impaired cholinergic function. In this study, the effects of a daily, 7-day, intraperitoneal administration of saffron on cognitive functions were examined in both healthy adult (4 months old) and aged (20 months old), male Balb-c mice (n=8/group), by passive avoidance test. Whole brain homogenates (minus cerebellum) were collected for examination of brain oxidative markers, caspase-3 and acetylcholinesterase (AChE) activity. Results showed that saffron-treated mice exhibited significant improvement in learning and memory, accompanied by reduced lipid peroxidation products, higher total brain antioxidant activity and reduced caspase-3 activity in both age groups of mice. Furthermore, salt- and detergent-soluble AChE activity was significantly decreased only in adult mice. Thus, we showed, for the first time, that the significant cognitive enhancement conferred by saffron administration in mice, is more closely related to the antioxidant reinforcement. Next, we compared the effect of saffron (1-250 μg/mL), crocetin and safranal (1-125 μM) on H(2)O(2)-induced toxicity in human neuroblastoma SH-SY5Y cells. Both saffron and crocetin provided strong protection in rescuing cell viability (MTT assay), repressing ROS production (DCF assay) and decreasing caspase-3 activation. These data, together with earlier studies suggest that crocetin is a unique and potent antioxidant, capable of mediating the in vivo effects of saffron.

Vaccination of Patients With Advanced Non–Small-Cell Lung Cancer With an Optimized Cryptic Human Telomerase Reverse Transcriptase Peptide

PURPOSE To evaluate the immunological and clinical response as well as the safety of the optimized peptide telomerase reverse transcriptase p572Y (TERT572Y) presented by HLA-A*0201 in patients with advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS Twenty-two patients with advanced NSCLC and residual (n = 8) or progressive disease (PD; n = 14) following chemotherapy and/or radiotherapy received two subcutaneous injections of the optimized TERT572Y peptide followed by four injections of the native TERT572 peptide administered every 3 weeks. Peptide-specific immune responses were monitored by enzyme-linked immunosorbent spot assay and/or TERT572Y pentamer staining. RESULTS Twelve (54.5%) of 22 patients completed the vaccination program. Toxicity consisted primarily of local skin reactions. TERT572-specific CD8+ cells were detected in 16 (76.2%) of 21 patients after the second vaccination, and 10 (90.9%) of 11 patients after the sixth vaccination. Stable disease (SD) occurred in eight (36.4%) of 22 vaccinated patients, with three (13.6%) having had PD before entering the study. The median duration of SD was 11.2 months. After a median follow-up of 10.0 months, patients with early developed immunological response (n = 16) had a significantly longer time to progression and overall survival (OS) than nonresponders (n = 5; log-rank tests P = .046 and P = .012, respectively). The estimated median OS was 30.0 months (range, 2.8 to 40.0 months) and 4.1 months (range, 2.4 to 10.9 months) for responders and nonresponders, respectively. CONCLUSION TERT572Y peptide vaccine is well tolerated and effective in eliciting a specific T cell immunity. Immunological response is associated with prolonged survival. These results are encouraging and warrant further evaluation in a randomized study.

The phosphodiesterase 5 inhibitor sildenafil stimulates angiogenesis through a protein kinase G/MAPK pathway

cGMP‐degrading pathways have received little attention in the context of angiogenesis. In the present study we set out to determine whether cGMP‐specific phosphodiesterase 5 (PDE5) inhibition affects new blood vessel growth. Incubation of chicken chorioallantoic membranes (CAMs) in vivo with sildenafil increased vascular length in a dose‐dependent manner. Moreover, incubation of cultured endothelial cells (ECs) with the PDE5 inhibitor promoted proliferation, migration, and organization into tube‐like structures. The effects of sildenafil on the angiogenesis‐related properties of EC could be blocked by pre‐treatment with the soluble guanylyl cyclase (sGC) inhibitor ODQ or the protein kinase G (PKG) I inhibitor DT‐3. In addition, over‐expression of sGC in EC led to an enhanced growth and migratory response to sildenafil. To study the signaling pathways implicated in the sildenafil‐stimulated angiogenic responses we determined the phosphorylation status of mitogen‐activated protein kinase (MAPK) members. Incubation of cells with sildenafil increased both extracellular signal regulated kinase 1/2 (ERK1/2) and p38 phosphorylation in a time‐dependent manner. Inhibition of MEK by PD98059 and p38 with SB203580 blocked sildenafil‐induced proliferation and migration, respectively, suggesting that these MAPK members are downstream of PDE5 and mediate the angiogenic effects of sildenafil. PDE5 inhibitors could, thus, be used in disease states where neo‐vessel growth is desired. J. Cell. Physiol. 211: 197–204, 2007.

Reactions of 3d-metal nitrates with N,N′-bis(2-pyridyl)urea (LH2): preparation, X-ray crystal structures and spectroscopic studies of the products trans-[M(II)(ONO2)2(LH2)2] (M=Mn, Fe, Co, Ni, Cu, Zn) and mer-[Co(III)(LH)2](NO3)·MeOH

Abstract The reactions of M(NO 3 ) 2 ·4H 2 O (M=Mn, Zn), M(NO 3 ) 2 ·6H 2 O (M=Co, Ni), Cu(NO 3 ) 2 ·3H 2 O and Fe(NO 3 ) 3 ·9H 2 O with the di-substituted urea ligand N , N ′-bis(2-pyridyl)urea (LH 2 ) were studied in methanol. The new complexes trans -[M(II)(ONO 2 ) 2 (LH 2 ) 2 ], where M=Mn ( 1 ), Fe ( 2 ), Co ( 4 ), Ni ( 6 ), Cu ( 7 ) and Zn ( 8 ), mer -[Co(III)(LH) 2 ](NO 3 )·MeOH ( 5 ) and [Fe 2 (III,III)O(NO 3 ) 4 (LH 2 ) 2 (MeOH) 2 ] ( 3 ) have been isolated. Of remarkable chemical interest is the reduction of iron(III) under aerobic conditions to give complex 2 and the presence of the deprotonated ligands in the cobalt(III) complex 5 . Complexes 1 , 2 and 4 – 8 have been structurally characterised by single-crystal X-ray studies. The structures of 1 , 2 , 4 and 6 – 8 consist of centrosymmetric octahedral molecules; the ligand LH 2 behaves as a bidentate chelate with the ligated atoms being the urea (amide) oxygen and one of the 2-pyridyl nitrogens, while the coordination sphere of the metal ion is completed by two monodentate nitrato groups. The coordination geometry around low-spin Co(III) in 5 is distorted octahedral; LH − chelates as a tridentate through both pyridyl nitrogen atoms and the deprotonated urea nitrogen atom forming one six- and one four-membered chelate ring. The new complexes were characterised by elemental analyses, thermal decomposition data, magnetic susceptibilities at room temperature and spectroscopic (IR, far-IR, Raman, UV–Vis, 57 Fe-Mossbauer, 1 H NMR) techniques. An oxo-bridged dinuclear structure with six-coordinate Fe(III) atoms is proposed for 3 . All data are discussed in terms of the nature of bonding and known or assigned structures.

Crocetin inhibits invasiveness of MDA-MB-231 breast cancer cells via downregulation of matrix metalloproteinases.

Crocetin is a carotenoid dicarboxylic acid which, in nature, is esterified with glucose or gentiobiose units forming the crocins, abundant components of saffron (a spice with many reputed medicinal uses). We have previously reported that saffron, crocins and crocetin inhibit breast cancer cell proliferation. In order to further study the effect of crocetin on breast cancer cells, we used the highly invasive MDA-MB-231 cells and measured the viability with the WST-1 assay and the invasiveness through a reconstituted basement membrane. After 24 h incubation, crocetin significantly inhibited not only proliferation but also invasion at 1 and 10 µM. Cancer invasiveness and metastasis are associated with the expression of matrix metalloproteinases (MMPs). In order to study the molecular changes of MMP expression that might accompany the observed crocetin effects, gene expression of MMPs was studied by RT-PCR, whereas protein expression and gelatinolytic activity were determined with Western blotting and zymography, respectively. The gene and protein expression of pro-MT1-MMP and pro-MT2-MMP were greatly attenuated by both crocetin and all- TRANS-retinoic acid (ATRA, used as control). Incubation with 10 µM crocetin for 24 h in serum-free conditions reduced pro-MMP-9 activity and pro-MMP-2/MMP-2 protein levels. When cultured in media with sera 2 and 5 %, crocetin at 10 μΜ also reduced gelatinase activity. The above findings show that crocetin, the main metabolite of crocins, inhibits MDA-MB-231 cell invasiveness via downregulation of MMP expression.

Diorganotin(IV) complexes of dipeptides containing the α-aminoisobutyryl residue (Aib): Preparation, structural characterization, antibacterial and antiproliferative activities of [(n-Bu)2Sn(H−1L)] (LH = H-Aib-L-Leu-OH, H-Aib-L-Ala-OH)

Two new organotin(IV) complexes with dianionic dipeptides containing the alpha-aminoisobutyryl residue (Aib) as ligands are described. The solid complexes [(n-Bu)(2)Sn(H(-1)L(A))] x 2MeOH (1 x 2MeOH) (L(A)H=H-Aib-L-Leu-OH) and [(n-Bu)(2)Sn(H(-1)L(B))] x MeOH (2 x MeOH) (L(B)H=H-Aib-L-Ala-OH) have been isolated and characterized by single-crystal X-ray crystallography and spectroscopic techniques (H(-1)L(2-) is the dianionic form of the corresponding dipeptide). Complexes 1 x 2MeOH and 2 x MeOH are monomeric with similar molecular structures. The doubly deprotonated dipeptide behaves as a N(amino), N(peptide), O(carboxylate) ligand and binds to the Sn(IV) atom. The five-coordinate metal ion has a distorted trigonal bipyramidal geometry. A different network of intermolecular hydrogen bonds in each compound results in very dissimilar supramolecular features. The IR, far-IR, Raman and (119)Sn NMR data are discussed in terms of the nature of bonding and known structures. The antibacterial and antiproliferative activities as well as the effect of the new compounds on pDNA were examined. Complexes 1 and 2 are active against the gram-positive bacteria Bacillus subtilis and Bacillus cereus. The IC(50) values reveal that the two compounds express promising cytotoxic activity in vitro against a series of cell lines.

99mTc-N4-[Tyr3]Octreotate Versus 99mTc-EDDA/HYNIC-[Tyr3]Octreotide: an intrapatient comparison of two novel Technetium-99m labeled tracers for somatostatin receptor scintigraphy.

Tetraamine-[Tyr3]octreotate (Demotate) is a somatostatin (SST) analogue that can be easily labeled with 99mTc at high specific activities and showed promising preclinical properties for SST receptor scintigraphy. This study reports on the first intra-patient comparison of 99mTc-Demotate and another 99mTc-labeled SST analogue, 99mTc-EDDA/HYNIC-TOC (HYNIC-TOC). Five patients with carcinoid tumors (n = 2) and endocrine pancreatic tumors (n = 3) were investigated with both radiopharmaceuticals. 99mTc-Demotate rapidly visualized somatostatin receptor positive tumors as early as 15 minutes post-injection (p.i.) with maximum tumor uptake and tumor/organ ratios already 1 hour p.i. Organs of predominant physiological uptake were the spleen and the kidneys with no intestinal excretion detectable up to 24 hours. 99mTc-Demotate exhibited faster pharmacokinetic properties compared to HYNIC-TOC. Tumor/organ ratios at equivalent time points were higher or comparable for 99mTc-Demotate in three patients with a matching scan result. Equivocal findings were observed in two patients, i.e. comparable uptake behavior in larger lesions with differences in smaller ones. 99mTc-Demotate is a promising agent for somatostatin receptor scintigraphy providing images of excellent quality as early as 1 hour after injection.

Helicobacter pylori neutrophil-activating protein activates neutrophils by its C-terminal region even without dodecamer formation, which is a prerequisite for DNA protection – novel approaches against Helicobacter pylori inflammation

Helicobacter pylori neutrophil‐activating protein (HP‐NAP) protects DNA from free radicals as a dodecamer through its ferroxidase activity without, however, directly binding to it. The retardation that was observed at pH 7.5 could be easily attributed to an iron effect, as it was revealed by experiments in the absence of HP‐NAP. A total loss of ferroxidase activity, dodecamer formation and DNA protection in environments rich in free radicals was observed after replacement of His25, His37, Asp52 and Lys134, which are located within the ferroxidase site, with Ala. Molecular dynamics simulations revealed that dimer formation is highly unlikely following mutation of the above amino acids, as the Fe2+ is no longer attracted with equal strength by both subunits. These findings probably indicate that iron plays an important role in the conformation of HP‐NAP by initiating the formation of stable dimers that are indispensable for the ensuing dodecamer structure. Very surprisingly, neutrophil activation appeared to be stimulated by structural elements that are localized within the C‐terminal region of both mutant HP‐NAP and wild‐type dodecamer HP‐NAP. In particular, the dodecamer conformation does not seem to be necessary for activation, and helices H3 (Leu69–Leu75) and H4 (Lys89–Leu114) or the linking coils (His63–Thr68 and Thr76–Ser88) are probably critical in stimulating neutrophil activation.

Structural features of angiotensin-I converting enzyme catalytic sites: conformational studies in solution, homology models and comparison with other zinc metallopeptidases.

Angiotensin-I Converting Enzyme (ACE) is a Zinc Metallopeptidase of which the three-dimensional structure was unknown until recently, when the X-ray structure of testis isoform (C-terminal domain of somatic) was determined. ACE plays an important role in the regulation of blood pressure due to its action in the frame of the Renin-Angiotensin System. Efforts for the specific inhibition of the catalytic function of this enzyme have been made on the basis of the X-ray structures of other enzymes with analogous efficacy in the hydrolytic cleavage of peptide substrate terminal fragments. Angiotensin-I Converting Enzyme bears the sequence and topology characteristics of the well-known gluzincins, a sub-family of zincins metallopeptidases and these similarities are exploited in order to reveal common structural elements among these enzymes. 3D homology models are also built using the X-ray structure of Thermolysin as template and peptide models that represent the amino acid sequence of the ACEs two catalytic, zinc-containing sites are designed and synthesized. Conformational analysis of the zinc-free and zinc-bound peptides through high resolution 1H NMR Spectroscopy provides new insights into the solution structure of ACE catalytic centers. Structural properties of these peptides could provide valuable information towards the design and preparation of new potent ACE inhibitors.