Plod kivija kao izvor alergena hrane

Since its first appearance on the market, kiwifruit has become very popular in the human diet due to its pleasant taste, low caloric value and high content of vitamin C. However, kiwifruit allergy has become a frequent cause of type I hypersensitivity in the western society. The molecular basis for kiwifruit allergy has been ascribed to up-to-now 11 identified IgE reactive molecules. They are proteins and glycoproteins with a molecular mass between 10 and 50 kDa. The major kiwifruit allergen is a cysteine protease denoted as Act d 1, which represents 50 % of the soluble protein extract. Due to differences in the abundance of the protein components and biological activity, the quality of kiwifruit extracts intended for allergy diagnosis can vary in content and amount of IgE reactive molecules. In addition, the quality of allergen extracts for allergy diagnosis depends on the fruit ripening stage and storage conditions. In terms of clinical reactivity, it has become evident that kiwifruit allergy is not a homogeneous disorder. Different patterns of IgE reactivity accompany several clinical subgroups that have been identified in different geographical regions. In the last decade, enormous progress has been made in the isolation and characterization of kiwifruit allergens. This paper presents an overview of the structural features of kiwifruit allergens.Od prvog pojavljivanja na tržištu plod kivija je postao izuzetno popularan sastojak humane ishrane usled prijatnog ukusa, niske kalorijske vrednosti i visokog sadržaja vitamina C. Međutim, alergija na kivi je postala učestali uzrok preosetljivosti tipa I u zapadnom društvu. Do sada je otkriveno 11 IgE vezujućih molekula koji čine molekulsku osnovu alergije na kivi. To su proteini i glikoproteini molekulskih masa između 10 i 50 kDa. Glavni alergen kivija je cistein-proteaza označena kao Act d 1, koja sačinjava 50 % rastvornih proteina ploda kivija. Usled razlike u zastupljenosti proteinskih komponenti i biološkoj aktivnosti, kvalitet proteinskih ekstrakata kivija koji se upotrebljavaju u dijagnostifikovanju alergije može varirati u sadržaju i količini IgE reaktivnih molekula. Takođe, kvalitet alergenih ekstrakata zavisi od stepena zrelosti voća prilikom branja, kao i od uslova skladištenja voća nakon branja. Po pitanju kliničke reaktivnosti postalo je očigledno da alergija na plod kivija ne predstavlja homogeni poremećaj. Različiti obrasci IgE reaktivnosti uočeni su kod nekolicine kliničkih podgrupa koje su identifikovane u različitim geografskim regijama. Tokom poslednje decenije načinjen je veliki napredak u izolovanju i karakterizaciji IgE vezujućih proteina kivija. U okviru ovog rada daćemo pregled strukturnih osobina alergenih proteina kivija

Nanobiokatalizatori za biogorivne ćelije i biosenzorne sisteme

This overview summarizes the application of enzymes in the manufacture and design of biofuel cells and biosensors. The emphasis will be put on the protein engineering techniques used for improving the properties of enzymes such as nanobiocatalysts, e.g. immobilization orientation, stability, activity and efficiency of electron transfer between immobilized enzymes and electrodes. Some possible applications in the military and some future designs of these electric devices will be discussed as well.U ovom preglednom članku je sumirana primena enzima u proizvodnji i dizajnu biogorivnih ćelija i biosenzora. Naglasak u pregledu literature je stavljen na tehnike proteinskog inžinjeringa, koje se koriste za poboljšanje osobina enzima u nanobiokatalizatorima kao što su orijentacija kod imobilizacije, stabilnost, aktivnost i efikasnost transfera elektrona između imobilizovanog enzima i elektrode. Na kraju pregleda je dato nekoliko primera moguće primene u vojsci. Nanobiokatalizatori su biokatalizatori u obliku enzima ili ćelija imobilizovani na nanomaterijalima. Koriste se kao sastavni elementi gorivnih ćelija u vidu imobilizovanih oksidoreduktaza na elektrodama. Na anodi se uz pomoć enzima oksiduju hemijska jedinjenja i elektroni predaju elektrodi, dok se na katodi elektroni uz pomoć druge oksidoreduktaze prebacuju sa elektrode na vodu ili kiseonik. Enzimi koji se koriste na anodi su glukoza oksidaza, formaldehid dehidrogenaza, alkohol dehidrogenaza i druge oksidaze šećera. Na katodi se uglavnom koriste lakaze, bilirubin oksidaza, peroksidaze i citohrom c oksidaza. Zahvaljujući razvoju nanotehnologije razvijaju se i minijaturne biogorivne ćelije koje proizvode električnu energiju za implantirane medicinske uređaje (insulinske pumpe, pejsmejkere, biosenzore) koristeći glukozu i kiseonik iz ljudske krvi. Biosenzori predstavljaju uređaje koji se sastoje iz biološke komponente, transducera i električne komponente. Oni pretvaraju koncen traciju hemijske supstance u električni signal i koriste se za analitiku. Kao biološka komponenta se mogu koristiti enzimi, monoklonska antitela, nukleinske kiseline i lipidi. Enzimska logička kola predstavljaju kombinaciju različitih biosenzora (enzimskih reakcija) koji mere nekoliko ulaznih parametara i na osnovu njih daju odgovarajući izlazni signal. Koristeći znanja kompjuterske tehnologije enzimskim logičkim kolima mogu se simulirati AND, OR, XOR, NOR, NAND, INHIB i XNOR logička kola. Za poboljšanje osobina biokatalizatora u cilju efikasnije primene u bioelektrokatalizi koriste se tehnike proteinskog inžinjeringa kao što su racionalni dizajn i dirigovana evolucija. Dirigovana evolucija koristi iterativne korake mutiranja i selekcije, kako bi biokatalizator evoluirao u pravcu koji nam je potreban. Najsporiji stupanj u ovoj tehnologiji predstavlja 'skrining', te se u novije vreme pomoću protočne citometrije i mikrofluidike pokušavaju razviti nove metode visoko propusnog skrininga. U literaturi opisani primeri dirigovane evolucije glukoza oksidaze, glukoza dehidrogenaze, formaldehid dehidrogenaze, laktat dehidrogenaze, peroksidaze i lakaze. Kombinacijom enzimskih logičkih kola i mikrofluidne tehnologije se pokušavaju napraviti laboratorije na čipu koje bi omogućile kontinuirano praćenje zdravstvenog stanja vojnika na bojnom polju i u slučaju šoka (ranjavanja) primenu odgovarajuće terapije u toku prvih 30 minuta od povrede. To bi obezbedilo veći stepen preživljavanja vojnika u ratu. Takođe upotrebom enzimskih logičkih kola i antitela moguće je postići uskladištenje i šifrovanje informacija, kao i zaštitu lozinkom, odgovarajućih elektronskih uređaja kao što su biogorivne ćelije Razvoj nanotehnologije, proteinskog inžinjeringa i molekularnog računarstva otvara vrata novim mogućnostima u proizvodnji i dizajnu biogorivnih ćelija i bisenzorskih sistema, kao i u skladištenju i zaštiti informacija

Supplementary data for article: Grozdanovic, M. M.; Drakulić, B. J.; Gavrović-Jankulović, M. Conformational Mobility of Active and E-64-Inhibited Actinidin. Biochimica et Biophysica Acta: General Subjects 2013, 1830 (10), 4790–4799. https://doi.org/10.1016/j.bbagen.2013.06.015

Supplementary material for: [https://doi.org/10.1016/j.bbagen.2013.06.015]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1398

Supplementary data for article: Blažić, M.; Kovačević, G.; Prodanović, O.; Ostafe, R.; Gavrović-Jankulović, M.; Fischer, R.; Prodanović, R. Yeast Surface Display for the Expression, Purification and Characterization of Wild-Type and B11 Mutant Glucose Oxidases. Protein Expression and Purification 2013, 89 (2), 175–180. https://doi.org/10.1016/j.pep.2013.03.014

Supplementary material for: [https://doi.org/10.1016/j.pep.2013.03.014]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1357

Pregled najčešće korišćenih metoda u karakterizaciji alergena

The characterization of an allergen is a troublesome and difficult process, as it requires both the precise biochemical characterization of a (glyco)protein molecule and the establishment of its susceptibility to IgE antibodies, as they are the main link to histamine release in some hypersensitivity states (type I allergies). As the characterization of an allergen includes molecular weight determination of the allergenic molecule, its structure determination, physicochemical properties, IgE binding properties of the allergen molecule, and its allergenicity, an overal review of which biochemical and immunochemical methods are used in achieving this goal are presented in this paper. The information on the molecular level on the stuctures of allergens indicates that allergens are considerably heterogeneous protein structures, and that there is no particular aminoacid sequence which is responsible for the allergenicity. Therefore, information gained from detailed structural, functional and immunochemical studies of these intriguing molecules, which nowadays modulate a variety of pathophysiological conditions, would greatly improve our understanding of the underlying disease mechanisms, and the way to handle them.Okarakterisati alergen je težak i mukotrpan zadatak, jer zahteva preciznu biohemijsku karakterizaciju (gliko)proteinskog molekula, kao i ustanovljavanje njegove sposobnosti da vezuje IgE, jer je to glavna spona ka oslobađanju histamina u nekim stanjima preosetljivosti (alergije tipa I). Karakterizacija alergena podrazumeva određivanje molekulske mase, određivanje strukture, fizikohemijskih svojstava, IgE vezujućih osobina i njegovu alergenost. Ovaj rad daje pregled koje se biohemijske i imunohemijske metode najčešće koriste radi postizanja tog cilja. Informacije koje su do sada dobijene o strukturi alergena pokazuju da su ovi molekuli izuzetno heterogene strukture i da ne postoji određena aminokiselinska sekvencija koja bi mogla da predvidi alergenost datog molekula. Međutim, informacije dobijene iz detaljnih strukturnih studija ovih molekula će doprineti našem razumevanju patofizioloških procesa koji su u osnovi alergijskih oboljenja i unaprediće način na koji ih tretiramo

Supplementary data for article: Grozdanović, M. M.; Burazer, L. M.; Gavrović-Jankulović, M. Kiwifruit (Actinidia Deliciosa) Extract Shows Potential as a Low-Cost and Efficient Milk-Clotting Agent. International Dairy Journal 2013, 32 (1), 46–52. https://doi.org/10.1016/j.idairyj.2013.03.001

Supplementary material for: [https://doi.org/10.1016/j.idairyj.2013.03.001]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/1589

Ispitivanje stabilnosti vakcine kućne prašine za sublingvalnu imunoterapiju

Allergen-specific immunotherapy with house dust mite (HDM) allergen extracts can effectively alleviate the symptoms of allergic rhinitis and asthma. The efficacy of the immunotherapeutic treatment is highly dependent on the quality of house dust mite vaccines. This study was performed to assess the stability of house dust mite allergen vaccines prepared for sublingual immunotherapy. Lyophilized Dermatophagoides pteronyssinus (Dpt) mite bodies were the starting material for the production of sublingual vaccines in four therapeutic concentrations. The stability of the extract for vaccine production, which was stored below 4 °C for one month, showed consistence in the protein profile in SDS PAGE. ELISA-inhibition showed that the potencies of Dpt vaccines during a 12 month period were to 65-80 % preserved at all analyzed therapeutic concentrations. This study showed that glycerinated Dpt vaccines stored at 4°C preserved their IgE-binding potential during a 12 month period, implying their suitability for sublingual immunotherapeutic treatment of HDM allergy.Alergen-specifična imunoterapija predstavlja postupak koji može da promeni tok bolesti kod alergijskog rinitisa i astme. Kvalitet vakcine pripremljene od kućnih grinja značajno utiče na efikasnost imunoterapeutskog tretmana. Ispitivanje je imalo za cilj procenu stabilnosti vakcine kućnih grinja namenjene za sublingvalnu imunoterapiju. Telo liofilozovanih Dermatophagoides pteronyssinus grinja (Dpt-grinja) upotrebljeno je kao polazni materijal za proizvodnju sublingvalne vakcine u 4 različita terapeutska razblaženja. Potentnost Dpt vakcina praćena ELISA-inhibicijom u 4 terapeutska razblaženja, nakon 12 meseci zadržan je u svim razblaženjima u opsegu 65-80%. Ispitivanje je pokazalo da glicerolom stabilizovane Dpt vakcine za subligvalnu imunoterapiju, uz čuvanje na 4°C zadržavaju alergeni potencijal ( gt 65%) nakon 12 meseci od njihove pripreme, i kao takve mogu se koristiti za tretman alergije na kućne grinje

Production, purification and structural characterisation of recombinant BanLec-Bet v 1

The sublingual route of allergens administration in allergen-specific immunotherapy (ASIT) is proven to be a successful way to treat patients with respiratory allergy. The trend of replacing natural extracts with purified recombinant allergens is growing. Although the purified allergens themselves are not good immunogens, the combined vector systems and adjuvans can improve their immunogenicity 1. Cell surfaces are decorated by different glycan structures, so the lectins specific for these glycans can be used to deliver particular therapeutic to target specific tissue 2. Banana lectin (BanLec) is mannose-specific protein which belongs to the subfamily of Jacalin related lectins 3. Apart from its characteristic to bind glycans, BanLec also modulates immune cells in vitro 4. On the other hand, Bet v 1 (Betula verrucosa) is the major birch pollen allergen. T-cell epitops are distributed over almost entire protein structure 5. In the study the recombinant BanLec-Bet v 1 construct is designed, produced by the recombinant DNA technology, purified and characterized by classical biochemical methods for the application in the ASIT of birch pollen allergy. The expression of newly designed BanLec-Bet v 1 was performed in E. coli BL21 (DE3). After expression the protein was found in the inclusion bodies from which it was extracted with 4 M urea solution. After renaturation, affinity chromatography (Sephadex G-75 superfine) was used for protein purification. Biochemical characterization of the chimera was performed by: SDS PAGE electrophoreses, CD spectroscopy and mass spectrometry. Biological activity of the construct was confirmed by binding of BanLecBet v 1 to a horseradish peroxidase glycoprotein in ELISA. Purified BanLec-Bet v 1 showed molecular mass of 32 kDa. CD spectra of the recombinant construct revealed well defined secondary structures with predominant beta sheets (41.2%). By mass spectrometry 51.8% of the BanLec-Bet v 1 primary structure was confirmed. Biologicaly active recombinant BanLec-Bet v 1 was produced by the recombinant DNA technology. Further in vitro and in vivo studies will evaluate immunomodulatory potential of BanLec-Bet v 1 for application in ASIT