92 research outputs found
Optimization of the heterologous expression of banana glucanase in Escherichia coli (vol 77, pg 43, 2012)
For the heterologous production of a banana glucanase in Escherichia coli, its gene (GenBank GQ268963) was cloned into a pGEX-4T expression vector as a fusion protein with glutathione-S-transferase (GST). BL21 cells transformed with the GST-Mus a 5 construct were employed for production of the protein induced by 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). The conditions for protein expression were optimized by varying the temperature (25, 30 and 37 C) and duration of protein expression (3, 6 and 12 h). The level of protein production was analyzed by densitometry of the sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG) after electrophoretic resolution of the respective cell lysates. The optimal protein expression for downstream processing was obtained after 12 h of cell growth at 25 degrees C upon addition of IPTG. Recombinant GST-Mus a 5 purified by glutathione affinity chromatography revealed a molecular mass of about 60 kDa. The IgE and IgG reactivity of the rGST-Mus a 5 was confirmed by dot blot analysis with sera of individual patients from subjects with banana allergy and polyclonal rabbit antibodies against banana extract, respectively. The purified recombinant glucanase is a potential candidate for banana allergy diagnosis.Za potrebe proizvodnje u Escherichia coli gen glukanaze iz banane (GenBank GQ268963) je ukloniran u ekspresioni vektor pGEX-4T sa glutation-S-transferazom (GST). Proizvodnja ovog proteina u Äelijama je indukovana 1 mM izopropil-Ī²-D-tiogalaktopiranozidom (IPTG). Uslovi za ekspresiju proteina su optimizovani variranjem temperature (25, 30 i 37Ā°C) i dužine trajanja proteinske sinteze (3, 6 i 12 h). Nivo proizvodnje proteina je analiziran denzitometrijom SDS-PA gela nakon elektroforetskog razdvajanja Äelijskih lizata. Optimalna proizvodnja proteina za njegovo dalje procesovanje je dobijena gajenjem Äelija nakon dodatka IPTG na 25Ā°C tokom 12 h. Rekombinantni GST-Mus a 5 preÄiÅ”Äen afinitetnom hromatografijom sa glutationom pokazuje molekulsku masu od 60 kDa. IgE i IgG reaktivnost izolovane glukanaze potvrÄena je u 'dot blot' sa pojedinaÄnim serumima osoba alergiÄnih na bananu, i sa poliklonskim zeÄijim antitelima na ekstrakt banane, redom. PreÄiÅ”Äena rekombinantna glukanaza je potencijalan kandidat za dijagnozu alergije na bananu
Optimizacija heterologe proizvodnje glukanaze iz banane u E. coli
For the heterologous production of a banana glucanase in Escherichia coli, its gene (GenBank GQ268963) was cloned into a pG EX-4T expression vector as a fusion protein with glutathione-S-transferase (GST). BL21 cells transformed with the GST-Mus a 5 con struct were employed for production of the protein induced by 1 mM isopropyl-Ī²-D-thiogalactopyranoside (IPTG). The conditions for protein expression were optimized by varying the temperature (25, 30 and 37Ā°C) and duration of protein expression (3, 6 and 12 h). The level of protein production was analyzed by densitometry of the sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG) after electrophoretic resolution of the respective cell lysates. The optimal protein expression for downstream processing was obtained after 12 h of cell growth at 25Ā°C upon addition of IPTG. Recombinant GST-Mus a 5 purified by glutathione affinity chromatography revealed a molecular mass of a bout 60 kDa. The IgE and IgG reactivity of the rGST-Mus a 5 was confirmed by dot blot an analysis with sera of individual patients from subjects with banana allergy and polyclonal rabbit antibodies against banana extract, respectively. The purified recombinant glucanase is a potential candidate for banana allergy diagnosis.Za potrebe proizvodnje u Escherichia coli gen glukanaze iz banane (GenBank GQ268963) je ukloniran u ekspresioni vektor pGEX-4T sa glutation-S-transferazom (GST). Proizvodnja ovog proteina u Äelijama je indukovana 1 mM izopropil-Ī²-D-tiogalaktopiranozidom (IPTG). Uslovi za ekspresiju proteina su optimizovani variranjem temperature (25, 30 i 37Ā°C) i dužine trajanja proteinske sinteze (3, 6 i 12 h). Nivo proizvodnje proteina je analiziran denzitometrijom SDS-PA gela nakon elektroforetskog razdvajanja Äelijskih lizata. Optimalna proizvodnja proteina za njegovo dalje procesovanje je dobijena gajenjem Äelija nakon dodatka IPTG na 25Ā°C tokom 12 h. Rekombinantni GST-Mus a 5 preÄiÅ”Äen afinitetnom hromatografijom sa glutationom pokazuje molekulsku masu od 60 kDa. IgE i IgG reaktivnost izolovane glukanaze potvrÄena je u 'dot blot' sa pojedinaÄnim serumima osoba alergiÄnih na bananu, i sa poliklonskim zeÄijim antitelima na ekstrakt banane, redom. PreÄiÅ”Äena rekombinantna glukanaza je potencijalan kandidat za dijagnozu alergije na bananu
Allergenic potency of kiwi fruit during fruit development
Food allergies, including kiwi fruit allergy, have been the subject of extensive research in the last few years. The aim of this study was to examine a possible relationship between the developmental stage of kiwi fruit and its allergenic potency. The protein and allergen patterns of kiwi fruit extracts in September, October, November and December fruit in the period from 2000-2002 were analysed. One of the factors that may contribute to the difficulties in proposing well-defined and standardized fruit extracts should also be the time of fruit harvesting. In this particular case, when the kiwi fruit was edible throughout November and December, we showed discrepancies in allergen content and potencies both in qualitative and quantitative terms. Two major allergens of kiwi fruit, Act c 1 and Act c 2, mainly accounted for the highest allergenic potential of November kiwi extract in vivo and in vitro. Not only the content of major allergens, but also the ratio of different proteins and even isoforms of the same allergen (Act c 2) change with fruit ripening. These findings should be taken into account during preparation of extracts for allergy diagnosis
Izolovanje i karakterizacija 68 kD alergena iz ekstrakta kuÄnih grinja
House dust mites (HDM) represent a major source of allergens, contributing to the increasing incidence of type I hypersensitivity disease worldwide. Over 30 different IgE-binding proteins from the HDM extract were detected. Although group 1 and 2 have been identified as major allergens, due to the safety and efficacy of allergy diagnosis and immunotherapy, there is a need to carefully evaluate the clinical relevance of other allergens present in the HDM extract. In regard to this, a high molecular mass allergen of about 68 kD was purified from the HDM extract using a combination of gel permeation chromatography and reversed-phase chromatography. The IgG and IgE reactivity of the purified protein were preserved during the purification process, as confirmed by Western blot analysis with polyclonal rabbit antibodies and dot blot analysis with a pool of sera from subjects with house dust mite allergy, respectively. In addition, the IgE reactivity was confirmed using ELISA testing with nine patient sera. The biological potency of the 68 kD allergen was confirmed by skin prick testing in five allergic subjects, suggesting that the high molecular mass allergen is a good candidate for component-resolved diagnosis of house dust mite allergy and eventual therapeutic treatment.Grinje iz kuÄne praÅ”ine predstavljaju jedan od glavnih izvora alergena koji su u znaÄajnoj meri doprineli porastu prvog tipa preosetljivosti. Preko 30 IgE-vezujuÄih proteina iz kuÄne praÅ”ine je detektovano do danas. Alergeni grupe 1 i 2 oznaÄeni su kao glavni alergeni kuÄne praÅ”ine. MeÄutim, da bi se poboljÅ”ala sigurnost i efikasnost dijagnoze i terapije alergijskih oboljenja izazvanih grinjama iz kuÄne praÅ”ine, neophodno je odrediti kliniÄki znaÄaj svih alergena iz ovog alergenskog izvora. U ovom radu izolovan je alergen visoke molekulske mase od 68 kD iz ekstrakta kuÄne praÅ”ine kombinovanjem gel-permeacione hromatografije i reversno-fazne hromatografije. IgG i IgE reaktivnost preÄiÅ”Äenog proteina je proverena u 'Western blot'-u i 'dot blot'-u sa poliklonskim zeÄijim antitelima na ekstrakt kuÄne praÅ”ine i 'pool'-om seruma osoba alergiÄnih na kuÄnu praÅ”inu, redom. 64 % pacijenata je pokazalo IgE reaktivnost na preÄiÅ”Äeni protein u ELISA testu. BioloÅ”ka reaktivnost preÄiÅ”Äenog alergena je potvrÄena u kožnim probama na pet pacijenata, ukazujuÄi da je preÄiÅ”Äen alergen dobar kandidat za dijagnozu alergije na kuÄnu praÅ”inu pojedinaÄnim komponentama i eventualni terapeutski tretman
Digestomics of Cow's Milk: Short Digestion-Resistant Peptides of Casein Form Functional Complexes by Aggregation
The aim of this study was to identify short digestion-resistant peptides (SDRPs) released by pepsin digestion of the whole cow's milk and examine their IgE reactivity and allergenicity. Raw milk was subjected to simulated gastric digestion. SDRPs were fractionated from the digests and identified by MS. Milk SDRPs were evaluated for aggregability, propensity to compete for IgE binding with individual milk allergens, and ability to bind IgG4 from allergic and milk-tolerant individuals. The majority of milk SDRPs originated from caseins (97% of peptides) and overlapped with the known IgE epitopes of cow's milk allergens. SDRPs competed with milk proteins for binding to human IgE and readily formed aggregates. The average peptide length was 10.6 +/- 3.5 amino acids. The ability to provoke allergenic in vivo responses was confirmed by skin-prick testing (SPT) in five milk-allergic subjects. This was attributed to the peptide ability to aggregate into non-covalent complexes. SDRPs are able to induce response in SPT, but only in 50% of the sera SDRPs were able to inhibit IgG4 binding to caseins. Hence, SDRPs corresponding to the mainly continuous epitopes of milk proteins induce allergenic in vivo responses in milk-allergic subjects due to aggregation
Izolovanje i karakterizacija 68 kD alergena iz ekstrakta kuÄnih grinja
House dust mites (HDM) represent a major source of allergens, contributing to the increasing incidence of type I hypersensitivity disease worldwide. Over 30 different IgE-binding proteins from the HDM extract were detected. Although group 1 and 2 have been identified as major allergens, due to the safety and efficacy of allergy diagnosis and immunotherapy, there is a need to carefully evaluate the clinical relevance of other allergens present in the HDM extract. In regard to this, a high molecular mass allergen of about 68 kD was purified from the HDM extract using a combination of gel permeation chromatography and reversed-phase chromatography. The IgG and IgE reactivity of the purified protein were preserved during the purification process, as confirmed by Western blot analysis with polyclonal rabbit antibodies and dot blot analysis with a pool of sera from subjects with house dust mite allergy, respectively. In addition, the IgE reactivity was confirmed using ELISA testing with nine patient sera. The biological potency of the 68 kD allergen was confirmed by skin prick testing in five allergic subjects, suggesting that the high molecular mass allergen is a good candidate for component-resolved diagnosis of house dust mite allergy and eventual therapeutic treatment.Grinje iz kuÄne praÅ”ine predstavljaju jedan od glavnih izvora alergena koji su u znaÄajnoj meri doprineli porastu prvog tipa preosetljivosti. Preko 30 IgE-vezujuÄih proteina iz kuÄne praÅ”ine je detektovano do danas. Alergeni grupe 1 i 2 oznaÄeni su kao glavni alergeni kuÄne praÅ”ine. MeÄutim, da bi se poboljÅ”ala sigurnost i efikasnost dijagnoze i terapije alergijskih oboljenja izazvanih grinjama iz kuÄne praÅ”ine, neophodno je odrediti kliniÄki znaÄaj svih alergena iz ovog alergenskog izvora. U ovom radu izolovan je alergen visoke molekulske mase od 68 kD iz ekstrakta kuÄne praÅ”ine kombinovanjem gel-permeacione hromatografije i reversno-fazne hromatografije. IgG i IgE reaktivnost preÄiÅ”Äenog proteina je proverena u 'Western blot'-u i 'dot blot'-u sa poliklonskim zeÄijim antitelima na ekstrakt kuÄne praÅ”ine i 'pool'-om seruma osoba alergiÄnih na kuÄnu praÅ”inu, redom. 64 % pacijenata je pokazalo IgE reaktivnost na preÄiÅ”Äeni protein u ELISA testu. BioloÅ”ka reaktivnost preÄiÅ”Äenog alergena je potvrÄena u kožnim probama na pet pacijenata, ukazujuÄi da je preÄiÅ”Äen alergen dobar kandidat za dijagnozu alergije na kuÄnu praÅ”inu pojedinaÄnim komponentama i eventualni terapeutski tretman
Rana alergijska reakcija na metilprednizolon sa tolerancijom drugih kortikosteroida
Introduction. In spite of the wide usage of corticosteroids for the treatment of a plethora of diseases, sometimes they can induce immediate hypersensitivity reactions, which are however uncommon. Case Outline. We report a case of immediate allergic reaction induced by intravenous methylprednisolone given before operation for surgical repair of an arm contracture as a sequel of burns, which the child had tolerated a month before. Six weeks later the patient repeated the anaphylactic reaction during skin testing to methylprednisolone. In addition, basophile activation test with methylprednisolone (BAT) was positive. Conclusion. This case report describes a patient who experienced intraoperative anaphylaxis and anaphylactic reaction induced by skin testing. This is the first report on induction of both anaphylactic reactions by methylprednisolone in the same child. Clinical findings, positive BAT and positive skin tests with methylprednisolone imply that the child developed type-I hypersensitivity. The lack of cross-reactivity with other corticosteroids emphasizes that the reactions were caused by the steroid molecule.Uvod. Uprkos Å”irokoj primeni kortikosteroida u leÄenju od razliÄitih bolesti, oni ponekad mogu izazvati ranu alergijsku reakciju. Prikaz bolesnika. Kod dvanaestogodiÅ”njeg deÄaka doÅ”lo je do rane alergijske reakcije izazvane intravenskom primenom metilprednizolona neposredno pre hirurÅ”ke intervencije, taÄnije, korekcije kontrakture Å”ake koja se javila kao komplikacija opekotine. Mesec dana pre pojave alergijske reakcije dete je primalo metilprednizolon i dobro ga podnosilo. Å est nedelja posle operacije ponovo se javila anafilaktiÄka reakcija tokom kožnog testiranja metilprednizolonom. Primenjen je i test aktivacije bazofila (BAT) ovim lekom, Äiji je nalaz bio pozitivan. ZakljuÄak. Ovo je prvi prikaz dve vrste anafilaktiÄke reakcije izazvane metilprednizolonom kod iste osobe. KliniÄka slika, pozitivni nalaz BAT i pozitivne kožne probe na metilprednizolon pokazuju da se kod deteta razvio prvi tip hipersenzitivne reakcije. Nedostatak unakrsne reaktivnosti s ostalim kortikosteroidima ukazuje na to da je alergijska reakcija izazvana steroidnim molekulom
Digestibilnost alergoida polena pelina u simuliranim uslovima gastrointestinalnog trakta
Chemically modified allergens (allergoids) have found use in both traditional and novel forms of immunotherapy of allergic disorders. Novel forms of immunotherapy include local allergen delivery, via the gastrointestinal tract. This study conveys the gastrointestinal stability of three types of mugwort pollen allergoids under simulated conditions of the gut. Allergoids of the pollen extract of Artemisia vulgaris were obtained by means of potassium cyanate, succinic and maleic anhydride. Gastrointestinal tract conditions (saliva, and gastric fluid) were simulated in accordance with the EU Pharmacopoeia. The biochemical and immunochemical properties of the derivatives following exposure to different conditions were monitored by determining the number of residual amino groups with 2,4,6-trinitrobenzenesulfonic acid, SDS PAGE, immunoblotting and inhibition of mugwort-specific IgE. Exposure to saliva fluid for 2 min did not influence the biochemical and immunochemical properties of the derivatives. In the very acidic conditions of the simulated gastric fluid, the degree of demaleylation and desuccinylation, even after 4 h exposure, was low, ranging from 10 to 30 %. The digestion patterns with pepsin proceeded rapidly in both the unmodified and modified samples. In all four cases, a highly resistant IgE-binding protein the Mwof which was about 28-35 kD, was present. Within the physiological conditions, no new IgE binding epitopes were revealed, as demonstrated by immunoblot and CAP inhibition of the mugwort specific IgE binding. An important conclusion of this study is the stability of the modified derivatives in the gastrointestinal tract of patients, within physiological conditions. The means that they are suitable for use in much higher concentrations in local forms of immunotherapy than unmodified ones.U ovom radu su prikazani rezultati ispitivanja stabilnosti tri tipa alergoida polena pelina u simuliranom želudaÄnom soku. KoristeÄi kalijum-cijanat anhidrid Äilibarne i anhidrid maleinske kiseline, napravljeni su alergoidi polena pelina (Artemisia vulgaris). Saliva i želudaÄni sok su simulirani na osnovu evropske farmakopeje. Biohemijske i imunohemijske osobine derivata posle izlaganja razliÄitim uslovima, praÄene su: odreÄivanjem broja slobodnih amino grupa u reakciji sa TNBS, SDS PAG elektroforezom, imunoblotom i odreÄivanjem pelin-specifiÄnog imunoglobulina E (IgE). Izlaganje salivi u trajanju od 2 minuta ne utiÄe na biohemijske i imunohemijske osobine derivata. U kiseloj sredini želudaÄnog soka ne dolazi do znaÄajnog demaleilovawa i desukcinilovanja. Äak i posle ÄetvoroÄasovnog izlaganja, taj procenat je u opsegu 10-30 %. Alergoidi pelina se trenutno digestuju pepsinom, sa izuzetkom visoko rezistentne proteinske trake molekulske mase 28-35 kD, koja odgovara važnom IgE-vezujuÄem proteinu polena pelina. Imunoblotom i CAP-inhibicijom je pokazano da, u okviru fizioloÅ”kih uslova, ne dolazi do stvaranja novih IgE-vezujuÄih epitopa. Hemijska stabilnost modifikovanih derivata u simuliranim uslovima želudaÄnog soka omoguÄuje da se tokom imunoterapije mogu primenjivati veÄe doze alergoida nego nemodifikovanog ekstrakta polena pelina
Digestomics of cow's milk: casein-derived digestion-resistant peptides aggregate into functional complexes
This paper discusses the improvements to flight simulators and their use in flight training
Supplementary data for the article: Stanic-Vucinic, D.; Stojadinovic, M.; Mirkov, I.; Apostolovic, D.; Burazer, L.; Atanaskovic-Markovic, M.; Kataranovski, M.; Cirkovic Velickovic, T. Hypoallergenic Acid-Sensitive Modification Preserves Major Mugwort Allergen Fold and Delivers Full Repertoire of MHC Class II-Binding Peptides during Endolysosomal Degradation. RSC Advances 2016, 6 (91), 88216ā88228. https://doi.org/10.1039/c6ra17261j
Supplementary material for: [https://doi.org/10.1039/c6ra17261j]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2321
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