Peptid markerek a fehérje allergia előrejelzésében = Peptide markers to predict food allergy

Az allergiás esetek száma világszerte növekszik és gyakran járványszerű méreteket is ölt. Az élelmiszerek közül a legtöbb megbetegedést a tojás, mogyoró, tej, diófélék, hal, kagyló és rák, búza és szója fogyasztása okozza érzékeny egyénekben. A pályázat keretében a búzafehérjék vizsgálatára proteomikai módszereket alkalmaztunk, ami a kétdimenziós elektroforézissel történő elválasztásból, a víz- és sóoldható fehérjék immun reaktivitásának detektálásából, és tömegspektroszkópiai - LC/MS és adatbázis alkalmazása - azonosításból állt. A búza víz- és sóoldható fehérjéi között összesen 13 allergén fehérjét - alfa-amiláz inhibitort, alfa-amiláz inhibitor prekurzort, 27 K fehérjét és egyéb fehérjét - azonosítottunk a 30 kDa-nál kisebb molekulatömegű tartományban. Az allergének epitopjainak megismerésén kívül fontos feladat különböző élelmiszerekből való detektálásuk. A pékasztmának nevezett betegségben szerepet játszó alfa-amiláz inhibitor család allergén fehérjéinek elválasztásához anioncserélő kromatográfiát, illetve szervetlen adszorbenst alkalmaztunk és a nagy alfa-amiláz inhibitor aktivitású frakciókat gyűjtöttük. Az inhibitorokban gazdag frakció gyors és egyszerű előállítása lehetővé teszi az allergén búzafehérjékből LC/MS eljárással kapott peptidtérkép alapján peptid markerek kiválasztását és ezek mérésével a búza allergének detektálását élelmiszerekben. | The number of allergic occurrence increases worldwide and often reaches epidemic proportions. Eggs, peanuts, milk, tree nuts, fish, shellfish, wheat and soy are sources of allergenic food proteins, the consumption of which leads to malfunctioning of the immune system in sensitive individuals. Within the framework of the application we applied proteomic approach, namely two-dimensional electrophoresis, immunoblotting, LC-MS/MS and databank accession, to identification of allergenic albumins/globulins of wheat. Of water- and salt soluble proteins of wheat, 13 have been identified - alfa-amylase inhibitors, alfa-amylase inhibitor precursors, 27 K protein, and others ? in the range of less than 30 kDa molecular mass. In addition to the explanation of the epitop structure, detection of allergens in various foodstuffs is a task of primary importance, too. To separate allergenic proteins of alfa-amylase inhibitor family, that cause bakers asthma, we used anion exchange chromatography as well as inorganic adsorbent, and fractions of high inhibitor activity were collected. A simple and quick preparation of inhibitor rich protein fraction makes it feasible to choose peptid markers on the basis of peptid mass mapping obtained by LC-MS/MS and accession to databank supply, by use of which detection of wheat allergens in food products is possible

Bulk and surface sensitivity of a resonant waveguide grating imager

We report the assessment of the sensitivity of a microplate-compatible resonant waveguide grating imager. The sensitivity to bulk refractive index changes was determined using a serial dilution of glycerol solution with the help of a refractometer. The surface sensitivity was examined using layer-by-layer polyelectrolyte films in conjunction with optical waveguide lightmode spectroscopy and characterized by the binding of acetazolamide to immobilized carbonic anhydrase under microfluidics. The results suggest that the imager has a limit of detection down to 2.2 × 10−6 for refractive index change and 0.078 ng/cm2 for the adsorbed mass. © 2014 AIP Publishing LL

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

Neuroprotective effects of repeated transient global ischemia and of kynurenine adminsitration induced by four-vessel occlusions on hippocampal CA1 neurons.

The hippocampal CA1 subfield is a brain region that is particularly sensitive to hypoxia. Although this subfield is selectively vulnerable to ischemic injuries manifested in delayed neuronal death (DND), the mechanism leading to neuronal degeneration is not fully understood. Burda recently reported that a second pathophysiological stress, applied within a suitable time, offers an opportunity for salvaging neurons in the CA1 region against DND (Neurochem. Res., 30: 1397-1405, 2005). In our study, NeuN immunohistochemistry was applied to detect survival CA1 neurons, while Fluoro-Jade B staining was used to evaluate the number of injured neurons after interventions resulting in transient global ischemia. Four groups of animals were used: 1: intact controls; 2: sham controls (2 vertebral arteries coagulated (2VAC), but 2 carotids sham-operated); 3: 2VAC + 2 carotids occluded (2CA) for 10 min; 4: 2VAC + 2CA (10 min) + 2 days later, a repeated 2CA (5 min). In group 3 (2VAC + 2CA (10 min)), marked cell destruction was found in the CA1 subfield: only 36.4% of the CA1 neurons survived. However, in group 4 (5-min second ischemic insult), the proportion of surviving cells in the CA1 region was 59.3%. There was no significant difference in CA1 cell loss between groups 1 and 2. Our findings suggest that the second ischemic stress, 2 days after the first ischemia induced by 2VAC + 2CA can be efficient in the prevention of DND. Neuroprotective effect was also found in four-vessel occlusion models after kynurenine (i.v.) administration

Az individuális sugárérzékenység szerepe és jelentősége a sugárterápiában = Role and significance of individual radiosensitivity in radiation oncology

Betegek 1. Bőr-biopsiás mintákból nyert fibroblast (Fb)-kultúrákban a survival fraction at 2 Gy (SF2)-értékek meghatározása 93 betegben. A radiogén második tumor relatív kockázatának (RR) becslése 6, 0-IA stádiumú, méhnyakrákos betegben. 2. A génexpresszió vizsgálata 7 normális, illetve 3 fokozottan sugárérzékeny sejtvonalban. 3. Multisegmentális (MS) besugárzás tanulmányozása 436 emlőrákbetegnél. Eredmények 1. Az akut dermatitis/mucositis 8/14 (57%), a reirradiáció 1/6, a késői idegrendszeri károsodás 4/11 (36%), a késői kötőszöveti/bőr toxicitás 9/50 (18%) beteg esetében járt együtt fokozott Fb-sugárérzékenységgel. A sugárkezelt, korai méhnyakrákos betegek RR-je rectumtumorra 9, anusrákra 23. 2. A normális sugárérzékenységű Fb-kből származó RNS-ekben a besugárzás hatására 109 gén expressziója növekedett, és 115 gén működése csökkent; fokozott sugárérzékenység esetén a hasonló adatok 142, illetve 56. 3. MS tervezéssel a tervezési céltérfogat (TCT) 90,9%-a esett a kívánatos dózisértékek közé (hagyományos tervezéssel: 82,8%), s a normális szervek dózisterhelése nem növekedett. Konklúziók 1. Nagyobb gyakorisággal alakulnak ki radiogén sérülések olyan betegekben, akik Fb-jének fokozott a sugárérzékenysége. 2. A sugárérzékenység sugárterápia előtti meghatározása elősegíthetné az egyénre szabott terápiás protokollok kialakítását. 3. Az MS sugárkezelés a normális szövetek dózis-terhelésének növekedése nélkül javítja a TCT dózisellátottságát. | Patients 1. Survival fractions at 2 Gy (SF2s) were determined using skin biopsy-based fibroblast (Fb) cultures in 93 pts. The relative risks (RRs) of radiogenic, secondary cancer were determined for 6, stage 0-IA, radiotherapy?treated cervix cancer pts. 2. Gene expressions were analyzed for 7 normal and 3 radiosensitive Fb cell lines. 3. Multisegmental (MS) radiotherapy was investigated in 436 breast cancer pts. Results 1. Inreased radiosensitivity was detected in 8/14 (57%) pts with acute dermatitis/mucositis, in 1/6 pts before reirradiation, in 4/11 (36%) pts with late neurological sequelae, and 9/50 (18%) pts with late subcutaneus/cutaneous toxicity. For radiotherapy-treated, early cervical cancer pts, the RRs are 9 and 23 to develope rectal and anal cancer, respectively. 2. Expressions of 109 genes were increased and 115 of them were decreased following irradiation of normal Fbs, while the comparative results for radiation-sensitive Fbs were 142 and 56, respectively. 3. Using MS radiotherapy, 90,9% of the planning target volme (PTV) was covered by the prescribed dose without an increase of radiation burden on normal tissues. Conclusions 1. The probability of radiation sequelae is higher among pts with radiosensitive Fbs. 2. Identification of radiosensitivity would help to determine individualized therapy protocols. 3. Introduction of MS radiotherapy would improve the PTV coverage without increasing the radiation burden on normal tissues

Development and In-Depth Characterization of Bacteria Repellent and Bacteria Adhesive Antibody-Coated Surfaces Using Optical Waveguide Biosensing

Bacteria repellent surfaces and antibody-based coatings for bacterial assays have shown a growing demand in the field of biosensors, and have crucial importance in the design of biomedical devices. However, in-depth investigations and comparisons of possible solutions are still missing. The optical waveguide lightmode spectroscopy (OWLS) technique offers label-free, non-invasive, in situ characterization of protein and bacterial adsorption. Moreover, it has excellent flexibility for testing various surface coatings. Here, we describe an OWLS-based method supporting the development of bacteria repellent surfaces and characterize the layer structures and affinities of different antibody-based coatings for bacterial assays. In order to test nonspecific binding blocking agents against bacteria, OWLS chips were coated with bovine serum albumin (BSA), I-block, PAcrAM-g-(PMOXA, NH(2), Si), (PAcrAM-P) and PLL-g-PEG (PP) (with different coating temperatures), and subsequent Escherichia coli adhesion was monitored. We found that the best performing blocking agents could inhibit bacterial adhesion from samples with bacteria concentrations of up to 10(7) cells/mL. Various immobilization methods were applied to graft a wide range of selected antibodies onto the biosensor’s surface. Simple physisorption, Mix&Go (AnteoBind) (MG) films, covalently immobilized protein A and avidin–biotin based surface chemistries were all fabricated and tested. The surface adsorbed mass densities of deposited antibodies were determined, and the biosensor;s kinetic data were evaluated to divine the possible orientations of the bacteria-capturing antibodies and determine the rate constants and footprints of the binding events. The development of affinity layers was supported by enzyme-linked immunosorbent assay (ELISA) measurements in order to test the bacteria binding capabilities of the antibodies. The best performance in the biosensor measurements was achieved by employing a polyclonal antibody in combination with protein A-based immobilization and PAcrAM-P blocking of nonspecific binding. Using this setting, a surface sensitivity of 70 cells/mm(2) was demonstrated

Development and In-Depth Characterization of Bacteria Repellent and Bacteria Adhesive Antibody-Coated Surfaces Using Optical Waveguide Biosensing

Bacteria repellent surfaces and antibody-based coatings for bacterial assays have shown a growing demand in the field of biosensors, and have crucial importance in the design of biomedical devices. However, in-depth investigations and comparisons of possible solutions are still missing. The optical waveguide lightmode spectroscopy (OWLS) technique offers label-free, non-invasive, in situ characterization of protein and bacterial adsorption. Moreover, it has excellent flexibility for testing various surface coatings. Here, we describe an OWLS-based method supporting the development of bacteria repellent surfaces and characterize the layer structures and affinities of different antibody-based coatings for bacterial assays. In order to test nonspecific binding blocking agents against bacteria, OWLS chips were coated with bovine serum albumin (BSA), I-block, PAcrAM-g-(PMOXA, NH2, Si), (PAcrAM-P) and PLL-g-PEG (PP) (with different coating temperatures), and subsequent Escherichia coli adhesion was monitored. We found that the best performing blocking agents could inhibit bacterial adhesion from samples with bacteria concentrations of up to 107 cells/mL. Various immobilization methods were applied to graft a wide range of selected antibodies onto the biosensor’s surface. Simple physisorption, Mix&Go (AnteoBind) (MG) films, covalently immobilized protein A and avidin–biotin based surface chemistries were all fabricated and tested. The surface adsorbed mass densities of deposited antibodies were determined, and the biosensor;s kinetic data were evaluated to divine the possible orientations of the bacteria-capturing antibodies and determine the rate constants and footprints of the binding events. The development of affinity layers was supported by enzyme-linked immunosorbent assay (ELISA) measurements in order to test the bacteria binding capabilities of the antibodies. The best performance in the biosensor measurements was achieved by employing a polyclonal antibody in combination with protein A-based immobilization and PAcrAM-P blocking of nonspecific binding. Using this setting, a surface sensitivity of 70 cells/mm2 was demonstrated

Integrin targeting of glyphosate and its cell adhesion modulation effects on osteoblastic MC3T3-E1 cells revealed by label-free optical biosensing

Abstract This study is a discovery of interesting and far reaching properties of the world leading herbicide active ingredient glyphosate. Here we demonstrate the cell adhesion-modifying characteristics of glyphosate affecting cellular interactions via Arg-Gly-Asp (RGD)-dependent integrins. This conclusion was supported by the observations that a glyphosate surface coating induced integrin-specific cell adhesion, while glyphosate in solution inhibited cell adhesion on an RGD-displaying surface. A sensitive, real-time, label-free, whole cell approach was used to monitor the cell adhesion kinetic processes with excellent data quality. The half maximal inhibitory concentration (IC50) for glyphosate was determined to be 0.47 ± 0.07% (20.6 mM) in serum-free conditions. A three-dimensional dissociation constant of 0.352 mM was calculated for the binding between RGD-specific integrins in intact MC3T3-E1 cells and soluble glyphosate by measuring its competition for RGD-motifs binding, while the affinity of those RGD-specific integrins to the RGD-motifs was 5.97 µM. The integrin-targeted affinity of glyphosate was proven using competitive binding assays to recombinant receptor αvβ3. The present study shows not only ligand-binding properties of glyphosate, but also illustrates its remarkable biomimetic power in the case of cell adhesion

Peripheral nerve injury influences the disinhibition induced by focal ischaemia in the rat motor cortex

Photothrombotic lesions were produced in the rat primary motor cortex, and the brain excitability was assessed in a paired-pulse stimulation protocol by transcranial recording, in parallel at 16 points of the frontal cortex, including the insulted and the surrounding areas. The cortical lesion reduced the inhibition in the extended frontal cortex, with a delay of a few minutes. Unilateral facial nerve transection, however, accelerated the widespread disinhibition. Although the mechanism is not clear in detail, both peripheral and central injury-induced disinhibition may have a significant impact on the recovery of the function