Az agy vérellátásának szabályozása újszülöttkori kísérletes hypoxiában = Regulation of cerebral blood supply following experimentally-induced hypoxia in the newborn

Altatott, újszülött malacokon vizsgáltuk a glutamáterg agyi vazododilatáció mechanizmusát. Megállapítottuk, hogy a kinurensav és a kvinolinsav, melyek a triptofán lebontási termékei befolyásolják az agyi erek NMDA és kainsav iránti érzékenységét. Malac agyból izolált arteriolákat vizsgálva arra a következtetésre jutottunk, hogy a glutamáterg vazodilatáció neuronális elemek közvetítésével jön létre. A mitochondriumok ATP szenzitív K+ csatornára ható diazoxide (DIAZ) segítségével bizonyítottuk, hogy agyi ischemiát követően az érendothelium és a neuronális-vaszkuláris csatolás károsodása mitochondriális eredetű. Patkányban igazoltuk, hogy a DIAZ előkezelés csökkenti az agyi ischemia következményeként létrejövő vér-agy gátkárosodást. Malac agyból nyert izolált mitochondriumokon demonstráltuk. DIAZ depolarizáló hatását, valamint az ezzel együtt jelentkező ROS termelődést. Szelektív enzim gátló szerek segítségével karakterizáltuk a ciklooxigenáz úton termelődő prosztanoidok szerepét az újszülöttkori agyi érreakciókban. Eredményeink szerint az asphyxiát követő 100% oxigénnel történő újraélesztés a hippocampusban és a kisagyban fokozza a neurodegenerációt. Mikrokeringési vizsgálataink rámutattak, hogy ennek lokális hemodinamikai okai lehetnek. Patkányban létrehozott krónikus agyi hipoperfúzió magatartási és morfológiai következményeinek vizsgálata során azt tapasztaltuk, hogy DIAZ előkezeléssel javíthatók a kognitív funkciók és csökken a hippocampális neuronális károsodás is. | In anesthetized newborn piglets we examined the mechanism of the glutamatergic vasodilation. We have shown that two metabolites of the kynurenine pathway of tryptophan degradation acting at the NMDA receptor, the antagonist kynurenic acid (KYNA) and the agonist quinolinic acid are capable of modulating the dilation of pial arterioles. In isolated piglet arteries, we demonstrated the lack of NMDA-induced vascular responses. We demonstrated that diazoxide (DIAZ), an activator of mitochondrial ATP-sensitive K+ (mitoKATP) channels, preserves endothelium-dependent cerebrovascular dilator responses after ischemia-reperfusion but does not affect the damage in the vascular smooth muscle. In a rat model of the cerebral ischemia we have shown that DIAZ pretreatment inhibited the edema formation and the disruption of the blood brain barrier. We characterized the effects of KATP channel openers and blockers on membrane potential and on the production of reactive oxygen species in isolated piglet mitochondria. Using selective enzyme inhibitors we further characterized the role of the cyclooxygenase (COX)-derived prostanoids in the cerebrovascular control of newborns. We demonstrated O2 toxicity after asphyxia was in the piglet hippocampus and cerebellum but not in the cerebral cortex or basal ganglia. The regional differences may be associated with haemodynamic factors. In a rat model of chronic hypoperfusion we demonstrated that pretreatment with DIAZ offers therapeutical prospects

Idegi plaszticitás és neuroprotekció lehetőségeinek kutatása triptofán metabolitokkal és származékaikkal = Neuronal plasticity and neuronal protection - research with triptophan-metabolites and conjugates

A pályázati támogatás olyan neuroprotekciós eljárások, anyagok kidolgozását szolgálta, mely ischemiás állapotok, traumák után fellépő hiperexcitáció csökkentésével mérsékli az ilyenkor fellépő másodlagos sejtpusztulást. A triptofán egyik metabolitja a kinurénsav (KYNA), mely az NMDA receptorok természetes inhibitoraként viselkedik a NMDA receptorokhoz kapcsolódva. A KYNA azonban nem megy át a vér-agy gáton, viszont előanyaga a kinurenin (KYN) igen. Kidolgoztunk egy módszert, melyben a KYN-t (300 mg/kg, i.p.) probeneciddel (PROB, 200 mg/kg, i.p.) együtt adva, oly mértékig meg tudjuk emelni a KYNA szintet az agyban, hogy az hatékonyan képes gátolni az agyi sérüléseket követő hiperexcitációt. Ezzel a módszerrel hatékony neuroprotekciót értünk el focalis és globalis agyi ischemiában, pentylentetrazollal (PTZ) kiváltott görcstevékenységben, de még migrén modellben is. Kifejlesztettünk két további kinurénsav származékot, a glukozamin-kinurénsavat (KYNA-NH-GLUC) és az SZR-72-nek nevezett szert, melyek szintén hatékony neuroprotektív anyagok, ugyanakkor átjutnak a vér-agy gáton, tehát szisztémásan is adhatók. Kísérleti körülmények között tehát hatékony neuroprotektív eljárást dolgoztunk ki, és hatékony anyagokat állítottunk elő. Továbbá, kimutattuk a KYNA-ról, hogy Janus-arcú, mert gátló hatása mellett, amit ?M-os koncentrációban fejt ki, ennél jóval alacsonyabb koncentrációban (200-250 nM) serkentő hatású. Mindez felveti a KYNA lehetséges neuromodulátor szerepét. | The aim of this project was to develop neuroprotective agents and protocols, which are able to reduce the late neuronal death following ischemic brain attack or brain trauma. The kynurenine pathway converts tryptophan into various compounds, including L-kynurenine (KYN), which in turn can be converted into the excitatory amino acid receptor (namely NMDA receptor) antagonist kynurenic acid (KYNA). The use of KYNA as a neuroprotective agent is rather restricted, however, because KYNA has only a very limited ability to cross the blood-brain barrier. In contrast, KYN crosses this barrier more readily. We have developed a protocol in which KYN (300 mg/kg, i.p.) administration together with probenedid (PROB, 200 mg/kg, i.p.), an organic acid transport blocker, led to significant neuroprotection in both focal and global brain ischemia, in traumatic brain attack, in migraine, and led to the inhibition of pentylenetetrazole (PTZ)-induced seizures. In addition, we have developed two kynurenic acid derivatives; glucosamine-kynurenic acid (KYNA-NH-GLUC) and SZR-72. Both of them cross the blood-brain barrier easily, and are neuroprotective agents. In addition to these experimental results, we have shown that KYNA is a Janus-faced agent, which in ?M concentration behaves as a neuroinhibitor, while in nM concentration, it stimulates the neuronal activity. This result suggests that KYNA might be play a role of a neuromodulator in the central nervoussystem

Membrane translocation of penetratin and its derivatives in different cell lines

The third helix of the homeodomain of the Antennapedia homeoprotein can translocate through the cell membrane into the nucleus and can be used as an intracellular vehicle for the delivery of oligopeptides and oligonucleotides. A 16-amino acid-long peptide fragment, called penetratin, is internalized by the cells in a specific, non-receptor-mediated manner. For a better understanding of the mechanism of the transfer, penetratin and two analogs were synthesized: (1) penetratin RQIKIWFQNRRMKWKK (peptide 1); (2) (6,14-Phe)-penetratin, RQIKIFFQNRRMKFKK (peptide 2); (3) dodecapeptide,RQIKIWF-R-KWKK (peptide 3); The conformation of penetratin peptides 1-3 was examined in both extracellular matrix-mimetic and membrane-mimetic environments. H-1-NMR and CD spectroscopic measurements were performed in mixtures of TFE/water with different ratios. Peptides 1-3 were labeled by reacting their N-terminal free amino group with fluorescein isothiocyanate (FITC). Membrane translocation of the labelled peptides was studied with cell cultures [WEHI 164 murine fibrosarcoma cells (WC/1); chicken fibroblast cells (CEC-32); chicken monocytic cells (HD-11); human fibroblast (SV 80) and human monocytic cells (MonoMac-6)]. Confocal laser scanning microscopy and flow cytometry assay were used to study membrane translocation. Amphiphilicity was calculated for each peptide. In our experiments all the penetratin peptides penetrated into the cells. Helical conformation and membrane translocation ability showed little correlation: substitution of the two Trp with Phe increased the stability of helical conformation but decreased membrane translocation activity. The results of fluorescence microscopy and flow cytometry show that penetratin can be translocated into the cells by two mechanisms: endocytosis and direct transport through the cell membrane. Copyright (C) 2003 John Wiley Sons, Ltd

Penetratin and Derivatives Acting as Antibacterial Agents

The synthesis, in vitro evaluation and conformational study of penetratin and structurally related derivatives acting as antibacterial agents are reported. Among the compounds evaluated here, two methionine sulphoxide derivatives (RQIKIWFQNRRM[O]KWKK-NH2 and RQIKIFFQNRRM[O]KFKK-NH2) exhibited the strongest antibacterial effect in this series. In order to better understand the antimicrobial activity obtained for these peptides, we performed an exhaustive conformational analysis using different approaches. Molecular dynamics simulations were performed using two different media (water and trifluoroethanol/water). The results of these theoretical calculations were corroborated using experimental CD measurements. The electronic study for these peptides was carried out using molecular electrostatic potentials obtained from RHF/6-31G(d) calculations. In addition, the non-apeptide RQIRRWWQR-NH2 showed strong inhibitory action against the Gram-negative and Gram-positive bacteria tested in this study

Peptidek és fehérjék, és peptid-nukleinsavak szintézise, szerkezetvizsgálata és biológiai szerepe = Synthesis, structure investigation and biological role of peptides, proteins and peptide nucleic acids

A neurodegeneratív betegségek kialakulásának jórészt közös mechanizmusa van: egy polipeptid vagy fehérje átalakul oldhatatlan aggregátummá, ez kicsapódik az idegsejtek között vagy a sejt belsejében és megakadályozza az ingerületvezetést, majd elpusztítja az idegsejteket. Alzheimer-kór modellkísérletekben igazoltuk, hogy a toxikus polipeptid (beta-amiloid 1-42) két fő aggregációs formája (Abeta-oligomer, ill. fibrillum) más receptorokon fejti ki hatását. A toxikus aggregátumok kialakulásában fontos szerepet játszhatnak a nehézfémionok, pl. a Zn2+, így a cink-kelátorok (pl. ACE-inhibitorok) az Alzheimer-kór potenciális gyógyszerei. Egy másik vegyületcsoport (peptidmimetikumok) úgy védi meg az idegsejteket, hogy beborítja az Abeta-aggregátumok felszínét és megakadályozza a kölcsönhatást a membránfehérjékkel (szabadalmi bejelentés). Gyógyszerek sejtbe jutását megkönnyítő, a sejtmembránon áthaladó peptid vektorokat terveztünk (penetratin származékok) és igazoltuk hatásmechanizmusukat (szabadalmi bejelentés). Egy humán beta-galaktozid kötő lektint (galektin-1) vizsgálva megállapítottuk, hogy igen hatásosan gátolja a gyulladásos és autoimmun folyamatokat, ez új immunoszupresszív stratégiát jelent. Az allergiás betegségek molekuláris hátterét vizsgálva a C3a szerkezetéből kiindulva olyan konformációsan gátolt peptidmimetikumokat állítottunk elő, amelyek megakadályozhatják a patológiás folyamatokat. Új szintézis módszereket dolgoztunk ki peptid nukleinsavak előállítására. | Most of the neurodegenerative diseases possess a common mechanism: a polypeptide or protein forms by misfolding water insoluble aggregates that precipitate extra- or intracellularly. These toxic aggregates hinder interneuronal connections and destroy neurons. Our laboratory proved in model experiments of Alzheimer's disease (AD) that the two main forms (oligomeric and fibrillar) of the toxic polypeptide beta-amyloid 1-42 affect different membrane receptors. Heavy metal ions, eng. Zn2+, may play key role in formation of toxic aggregates, therefore Zn-chelator drugs (e.g. ACE-inhibitors) could be used for the prevention of AD. Another group of compounds (peptides and peptidomimetics) protects neurons by covering the surface of Abeta-aggregates so preventing the interaction with membrane receptors (patent application). Novel peptide vector molecules (penetratin derivatives) were synthesized and studied, their mechanism of action was proven (patent application). Studying a human beta-galactoside binding lectin (galectin 1) it was proven, that the compound very effectively prevent inflammation and autoimmun processes providing a new strategy for immunosupression. The molecular background of allergic diseases was studied. Starting from the structure of the complement protein C3a, conformationally restricted peptidomimetics were synthesized that may prevent the pathological processes. Novel synthetic methods were introduced for the preparation of peptide nucleic acids

A nuclear import inhibitory peptide ameliorates the severity of cholecystokinin-induced acute pancreatitis

AIM: To assess the effect of our novel cell-permeable nuclear factor-kappaB (NF-kappaB) inhibitor peptide PN50 in an experimental model of acute pancreatitis. PN50 was produced by conjugating the cell-penetrating penetratin peptide with the nuclear localization signal of the NF-kappaB p50 subunit. METHODS: Pancreatitis was induced in male Wistar rats by administering 2X100 mug/kg body weight of cholecystokinin-octapeptide (CCK) intraperitoneally (IP) at an interval of 1 h. PN50-treated animals received 1 mg/kg of PN50 IP 30 min before or after the CCK injections. The animals were sacrificed 4 h after the first injection of CCK. RESULTS: All the examined laboratory (the pancreatic weight/body weight ratio, serum amylase activity, pancreatic levels of TNF-alpha and IL-6, degree of lipid peroxidation, reduced glutathione levels, NF-kappaB binding activity, pancreatic and lung myeloperoxidase activity) and morphological parameters of the disease were improved before and after treatment with the PN50 peptide. According to the histological findings, PN50 protected the animals against acute pancreatitis by favoring the induction of apoptotic, as opposed to necrotic acinar cell death associated with severe acute pancreatitis. CONCLUSION: Our study implies that reversible inhibitors of stress-responsive transcription factors like NF-kappaB might be clinically useful for the suppression of the severity of acute pancreatitis

Systemically administered glucosamine-kynurenic acid, but not pure kynurenic acid, is effective in decreasing the evoked activity in area CA1 of the rat hippocampus

The metabolism of tryptophan along the kynurenine pathway yields several neuroactive intermediates, including kynurenic acid, which is one of the few known endogenous N-methyl-D-aspartate receptor inhibitors; in parallel with this, it is an alpha 7 nicotinic acetylcholinergic receptor antagonist. On the basis of these properties, kynurenic acid might therefore come into consideration as a therapeutic agent in certain neurobiological disorders. However, the use of kynurenic acid as a neuroprotective agent is practically excluded because kynurenic acid hardly crosses the blood-brain barrier. We recently synthetized a new compound, glucosamine-kynurenic acid, which is presumed to cross the blood-brain barrier more easily. In this study, the effects of systemically administered kynurenic acid and glucosamine-kynurenic acid on CA3 stimulation-evoked population spike activity in region CA1 of the rat hippocampus were compared. The effect of kynurenic acid or glucosamine-kynurenic acid was augmented by probenecid (200 mg/kg), which inhibits kynurenic acid excretion from the cerebrospinal fluid. The results showed that, while kynurenic acid administered i.p. or i.v. in doses of 17, 34, 68 or 136 mu mol/kg did not cause any observable change in the animals, 136 mu mol/kg glucosamine-kynurenic acid (either i.p. or i.v.) resulted in the sudden death of all the animals. The dose of 68 mu mol/kg i.v., but not i.p., resulted in a sudden stoppage of breath, but the animals could be reanimated. As small a dose of glucosamine-kynurenic acid as 17 mu mol/kg i.p. resulted in a reduction in population spike amplitudes; this effect was further augmented by probenecid, whereas neither 17 mu mol/kg nor higher doses of pure kynurenic acid had a similar effect. The results presented here suggest that glucosamine-kynurenic acid passes the blood-brain barrier much more readily than does kynurenic acid. (c) 2005 Elsevier B.V. All rights reserved