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

Izolovanje funkcionalne ukupne RNK iz lišća i polena lipe (Tilia cordata)

The conditions required for the isolation of high quality total RNA from European linden (Tilia cordata) leaves and pollen were determined. Pure total RNA was isolated from linden leaves utilizing a Qiagen plant mini kit, while the total RNA isolated from linden pollen using this method was degraded. Successful isolation of total RNA from both linden pollen and leaves, however, was achieved following TRIzol (TM) preparation of the total RNA. The total RNA isolated using TRIzol (TM) was contaminated with genomic DNA but treatment with the enzyme DNase, in solution or on-column, efficiently removed the genomic DNA. Furthermore, the conditions for the elimination of genomic DNA contamination on-column and isolation of pure total RNA from leaves were optimized. The isolated total RNA from both leaves and pollen was used successfully in first-and second-strand cDNA synthesis reactions and in a reverse transcription polymerase chain reaction (RT-PCR), demonstrating that the total RNA isolated using this method was functional. In conclusion, pure and functional total RNA from T. cordata leaves and pollen (27.8 +/- 7.9 mu g g(-1) leaves; 25.7 +/- 1.1 mu g g(-1) pollen) could be obtained and was suitable for application in further molecular biology studies.Uspostavljeni su uslovi za izolovanje ukupne RNK iz lišća i polena evropske lipe (Tilia cordata). Korišćenjem komercijalno dostupnog pribora za izolovanje RNK iz biljaka izolovana je čista ukupna RNK iz lišća lipe, dok je korišćenjem iste metode dobijena degradirana RNK iz polena lipe. Uspešno izolovanje RNK iz lišća i polena je dobijeno korišćenjem TRIzol reagensa. RNK izolovana ovim metodom je kontaminirana genomskom DNK, koja je uspešno eliminisana korišćenjem enzima DNaze. Dalje su optimizovani i uslovi uklanjanja genomske DNK pomoću DNaze. Izolovana ukupna RNK iz oba izvora je dalje uspešno iskorišćena za sintezu prvog i drugog lanca klonske DNK, kao i u reverzno-transkriptivnoj PCR reakciji, dokazujući time da je korišćenjem ovog metoda izolovana funkcionalna ukupna RNK. U zaključku, dobijena je čista i funkcionalna RNK iz lišća i polena T. cordata (27,8±7,9 μg g-1 lišća; 25,7±1,1 μg g-1 polena) koja se može koristiti u daljim molekularno-biološkim istraživanjima

Proizvodnja lipaze iz Pseudozyma aphidis i utvrđivanje aktivnosti i stabilnosti lipaze u polarnim organskim rastvaračima

The production of lipase from Pseudozyma aphidis (DSM 70725) was determined in six different media. The highest lipase production was observed in a medium with glucose as the sole carbon source, and yeast extract and sodium nitrate as the nitrogen sources. The time course studies of growth and lipase production in the optimal medium revealed that the highest lipase production was achieved at the end of the log phase of growth, reaching the value of 35.0 U cm(-3) in the fifth day of cultivation. The effects of various polar, water-miscible, organic solvents on the activity and stability of the crude lipase produced by P. aphidis were evaluated. The hydrolytic activity of the crude lipase towards p-nitrophenyl palmitate (p-NPP) in aqueous media and in organic solvents was determined, using the same spectrophotometric assay in both the aqueous and organic media. The crude lipase preparation exhibited activity towards p-NPP only in acetone and acetonitrile, while the lipase was stable only in acetone, with 23 % residual activity after 24 h of incubation. These results suggested that lipase from P. aphidis can be used as a biocatalyst for potential applications in such organic solvents.Proizvodnja lipaze iz Pseudozyma aphidis utvrđena je u šest različitih medijuma. Najviša proizvodnja uočena je u medijumu gde je glukoza bila izvor ugljenika, a ekstrakt kvasca i natrijum-nitrat izvori azota. Praćenjem dinamike rasta i proizvodnje lipaze u optimalnom medijumu, uočeno je da se najviša proizvodnja lipaze dostiže pred kraj logaritamske faze rasta, i dostiže vrednost od 35 U cm-3 u petom danu kultivacije, što je četri puta veća proizvodnja od one do sada prijavljene u literaturi. Utvrđen je efekat različitih polarnih organskih rastvarača, mešljivih sa vodom, na aktivnost i stabilnost lipaze iz P. aphidis. Hidrolitička aktivnost lipaze prema para-nitrofenil-palmitatu (p-NPP-u) u vo- denoj sredini i organskim rastvaračima utvrđena je upotrebom istog spektrofotometrijskog testa. Pokazano je da lipaza ima aktivnost prema p-NPP-u samo u acetonu i acetonitrilu, dok je enzim stabilan jedino u acetonu i zadržava 23% aktivnosti nakon 24 časa inkubacije. Dobijeni rezultati ukazuju da lipaza iz P. aphidis može biti korišćena kao biokatalizator za potencijalne primene u acetonu kao medijumu

Isolation of functional total RNA from Tilia cordata leaves and pollen

The conditions required for the isolation of high quality total RNA from European linden (Tilia cordata) leaves and pollen were determined. Pure total RNA was isolated from linden leaves utilizing a Qiagen plant mini kit, while\ud the total RNA isolated from linden pollen using this method was degraded. Successful isolation of total RNA from both linden pollen and leaves, however, was achieved following TRIzol™ preparation of the total RNA. The total RNA isolated using TRIzol™ was contaminated with genomic DNA but treatment with the enzyme DNase, in solution or on-column, efficiently removed the genomic DNA. Furthermore, the conditions for the elimination of genomic DNA contamination on-column and isolation of pure total RNA from leaves were optimized. The isolated total RNA from both leaves and pollen was used successfully in first- and second-strand cDNA synthesis reactions and in a reverse transcription polymerase chain reaction (RT-PCR), demonstrating that the total RNA isolated using this method was functional. In conclusion, pure and functional total RNA from T. cordata leaves and pollen (27.8±7.9 μg g-1 leaves; 25.7±1.1 μg g-1 pollen) could be obtained and was suitable for application in further molecular biology studies

Stabilizacija α-glukozidaze u organskim rastvaračima imobilizacijom na makroporoznim (poli) glicidil metakrilatima različitih površinskih karakteristika

alpha-Glucosidase from baker's yeast was immobilized on macroporous copolymers of ethylene glycol dimethacrylate and glycidyl methacrylate, poly(GMA-co-EGDMA), with various surface characteristics and pore sizes ranging from 44 nm to 270 nm. Immobilization was done by glutaraldehyde on the copolymer previously modified with 1,2-diaminoethane. The specific activity of the obtained immobilized enzyme varied from 27 to 81 U/g depending on the employed copolymer. The half lives of the immobilized enzyme in cosolvents were influenced by the surface characteristics of the copolymer, ranging from 60 to 150 min in 35 % methanol and from 10 to 44 min in 45 % dimethyl sulphoxide (DMSO). The best stabilities were obtained when the enzyme was immobilized onto a copolymer having a pore size of 48 rim in methanol and 270 nm in DMSO.α-Glukozidaza izolovana iz pekarskog kvasca je imobilizovana na makroporoznim glicidil-metakrilatima različitih površinskih karakteristika i veličina pora od 44 do 270 nm. Imobilizacija je izvedena glutaraldehidom na polimeru prethodno modifikovanom sa 1,2-diaminoetanom. Specifična aktivnost dobijenog imobilizovanog enzima je varirala od 27 do 81U/g u zavisnosti od vrste korišćenog polimera. Poluživoti imobilizovanog enzima u korastvaračima su zavisili od površinskih karakteristika polimera i kretali su se u opsegu od 60 do 150 min u 35%(v/v) metanolu i od 10 do 44 min u 45 % (v/v) dimetilsufoksidu. Najveća stabilnost u metanolu je dobijena imobilizacijom enzima na polimeru sa veličinom pora od 48 nm a u dimetilsulfoksidu na polimeru sa veličinom pora od 270 nm

Digestibilnost β-laktoglobulina nakon njegovog unakrsnog povezivanja dejstvom lakaze iz Trametes versicolor i polifenola iz jabuke

beta-Lactoglobulin (BLG) is an important nutrient of dairy products and an important allergen in cow's milk allergy. The aim of this study was to investigate the potential of laccase to cross-link BLG in the presence of an apple phenolic extract (APE) and to characterize the obtained products for their digestibility by pepsin and pancreatin. The composition of the apple phenolics used for cross-linking was determined by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The apple phenolic extract contained significant amounts of quercetin glycosides, catechins and chlorogenic acid. The laccase cross-linked BLG in the presence of apple phenolics. The polymerization rendered the protein insoluble in the reaction mixture. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the cross-linking reaction mixture revealed a heterogeneous mixture of high molecular masses (cross-linked BLG), with a fraction of the BLG remaining monomeric. Enzymatic processing of BLG by laccase and apple polyphenols as mediators can decrease the biphasal pepsin pancreatin digestibility of the monomeric and cross-linked protein, thus decreasing its nutritional value. In addition, reduced BLG digestibility can decrease its allergenic potential. Apple polyphenols can find usage in the creation of new, more functional food products, designed to prevent obesity and hypersensitivity-related disorders.β-Laktoglobulin (BLG) je važan nutrijent mlečnih proizvoda i važan alergen kod alergija na kravlje mleko. Cilj ove studije je bilo ispitivanje potencijala lakaze da unakrsno poveže BLG u prisustvu fenolnog ekstrakta jabuke (APE), kao i karakterizacija dobijenih proizvoda sa aspekta njihove digestibilnosti pepsinom i pankreatinom. Kompozicija fenola jabuke korišćenih za unakrsno povezivanje određena je pomoću LC-ESI-MS. Fenolni ekstrakt jabuke sadrži znatne količine glikozida kvercetina, katehine i hlorogensku kiselinu. BLG je unakrsno povezan lakazom u prisustvu fenola jabuke, pri čemu je polimerizacija učinila BLG nerastvornim u reakcionoj smeši. SDS-PAGE analiza pokazala je da reakciona smeša sadrži heterogenu smešu velikih molekulskih masa (unakrsno povezan BLG), kao i deo zaostalog monomernog BLG. Enzimsko procesovanje BLG lakazom, u prisustvu polifenola jabuke kao medijatora, može smanjiti bifaznu pepsin-pankreatinsku digestibilnost kako monomernog, tako i unakrsno povezanog BLG, i na taj način smanjiti njegovu nutritivnu vrednost. Takođe, smanjena digestibilnost BLG može smanjiti njegov alergeni potencijal. Polifenoli jabuke mogu se koristiti za kreiranje novih, funkcionalnijih prehrambenih proizvoda, dizajniranih za prevenciju kako gojaznosti, tako i bolesti vezanih za preosetljivost

Standardi za proces nastave i učenja hemije

In this paper, there are frames given about making standards for the process of teaching and learning chemistry in our country. Standards of education in natural sciences represent criteria for students’ achievement assessment, for assessing if the quality and teaching allow students to learn natural sciences, for assessing professional praxis, for assessing quality of praxis and evaluation of educational policy. Development of standards in the field of natural sciences in the world is followed by these principles: natural sciences for all students, learning natural sciences is an active process, natural sciences at school reflect intercultural and cultural tradition which characterizes praxis in contemporary science, improvement of education in the field of natural sciences is a part of the systematic reform of education. Also, in this paper there is methodology suggested for development of standards for the process of teaching and learning chemistry in our country. .U ovom radu dati su okviri za donošenje standarda za proces nastave i učenja hemije u našoj zemlji. Standardi za obrazovanje u prirodnim naukama predstavljaju kriterijume za procenu učeničkih postignuća, za procenu da li kvalitet programa i nastave omogućava učenicima učenje prirodnih nauka, za procenu kvaliteta prakse vrednovanja i obrazovne politike. Razvoj standarda u oblasti prirodnih nauka u svetu praćen je sledećim principima: prirodne nauke su za sve učenike, učenje prirodnih nauka je aktivan proces, prirodne nauke u školi reflektuju intelektualnu i kulturnu tradiciju koja karakteriše praksu u savremenoj nauci, unapređenje obrazovanja u oblasti prirodnih nauka deo je sistematske reforme obrazovanja. Takođe, u radu je predložena metodologija za razvoj standarda za proces nastave i učenja hemije kod nas.

Izolovanje i karakterizacija 68 kD alergena iz ekstrakta kućnih 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 kućne prašine predstavljaju jedan od glavnih izvora alergena koji su u značajnoj meri doprineli porastu prvog tipa preosetljivosti. Preko 30 IgE-vezujućih proteina iz kućne prašine je detektovano do danas. Alergeni grupe 1 i 2 označeni su kao glavni alergeni kućne prašine. Međutim, da bi se poboljšala sigurnost i efikasnost dijagnoze i terapije alergijskih oboljenja izazvanih grinjama iz kućne prašine, neophodno je odrediti klinički značaj svih alergena iz ovog alergenskog izvora. U ovom radu izolovan je alergen visoke molekulske mase od 68 kD iz ekstrakta kućne prašine kombinovanjem gel-permeacione hromatografije i reversno-fazne hromatografije. IgG i IgE reaktivnost prečišćenog proteina je proverena u 'Western blot'-u i 'dot blot'-u sa poliklonskim zečijim antitelima na ekstrakt kućne prašine i 'pool'-om seruma osoba alergičnih na kućnu prašinu, redom. 64 % pacijenata je pokazalo IgE reaktivnost na prečišćeni protein u ELISA testu. Biološka reaktivnost prečišćenog alergena je potvrđena u kožnim probama na pet pacijenata, ukazujući da je prečišćen alergen dobar kandidat za dijagnozu alergije na kućnu prašinu pojedinačnim komponentama i eventualni terapeutski tretman

Standardi za proces nastave i učenja hemije

In this paper, there are frames given about making standards for the process of teaching and learning chemistry in our country. Standards of education in natural sciences represent criteria for students’ achievement assessment, for assessing if the quality and teaching allow students to learn natural sciences, for assessing professional praxis, for assessing quality of praxis and evaluation of educational policy. Development of standards in the field of natural sciences in the world is followed by these principles: natural sciences for all students, learning natural sciences is an active process, natural sciences at school reflect intercultural and cultural tradition which characterizes praxis in contemporary science, improvement of education in the field of natural sciences is a part of the systematic reform of education. Also, in this paper there is methodology suggested for development of standards for the process of teaching and learning chemistry in our country. .U ovom radu dati su okviri za donošenje standarda za proces nastave i učenja hemije u našoj zemlji. Standardi za obrazovanje u prirodnim naukama predstavljaju kriterijume za procenu učeničkih postignuća, za procenu da li kvalitet programa i nastave omogućava učenicima učenje prirodnih nauka, za procenu kvaliteta prakse vrednovanja i obrazovne politike. Razvoj standarda u oblasti prirodnih nauka u svetu praćen je sledećim principima: prirodne nauke su za sve učenike, učenje prirodnih nauka je aktivan proces, prirodne nauke u školi reflektuju intelektualnu i kulturnu tradiciju koja karakteriše praksu u savremenoj nauci, unapređenje obrazovanja u oblasti prirodnih nauka deo je sistematske reforme obrazovanja. Takođe, u radu je predložena metodologija za razvoj standarda za proces nastave i učenja hemije kod nas.

Parcijalna karakterizacija antibakterijskog peptida koji proizvodi probiotska bakterija Lactobacillus plantarum G2

The aim of this study was the partial characterization of the antimicrobial peptide bacteriocin G, produced by probiotic bacteria Lactobacillus plantarum G(2), which was isolated from a clinical sample of a healthy person. Antimicrobial substance was secreted in the supernatant of an L. plantarum G(2) culture, and showed a diverse spectrum of antimicrobial activity of all the tested strains of the genera Lactobacillus and the pathogenic bacteria Staphylococcus aureus and Salmonella abony. Isoclectric focusing revealed that bacteriocin G(2) is a cationic peptide (pI about 10) with a molecular mass of 2.2 kDa according to tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis, SDS-PAGE. The antimicrobial activity of bactcriocin G(2) was diminished by the proteolytic action of trypsin and proteinase K. Bacteriocin G(2) preserved its biological activity in the temperature range 40-60 degrees C (15 min), which was lost at 80 degrees C. Bacteriocin G(2) was stable in the pH range 2-9, while treatment with 1 % Tween 80 and 1 % urea resulted in increased antimicrobial activity. The probiotic strain L. plantarum G(2) produces the antimicrobial substance proteinaceous in nature with bacteriocin characteristics. Bacteriocin production is one of the key properties of probiotic bacteria with clinical potential as anti-infective agents, which will increase the likelihood of its in vivo efficacy.Cilj ovog istraživanja je bila parcijalna karakterizacija antimikrobnog peptida iz probiotske bakterija Lactobacillus plantarum G2 izolovane iz kliničkog uzorka zdrave osobe. Antimikrobno jedinjenje iz L. plantarum G2, označeno kao bakteriocin G2, dobijeno iz supernatanta bakterijske kulture, pokazalo je širok spektar antimikrobne aktivnosti, inhibirajići rast svih ispitivanh vrsta roda Lactobacillus, kao i patogenih bakterija Staphylococcus aureus i Salmonella abony. Bakteriocin G2 je osetljiv na proteolitičko dejstvo tripsina i proteinaze K. Antimikrobna aktivnost je stabilna u opsegu 40-60°C (15 min), ali se gubi na temperaturi od 80°C. Ustanovljeno je da je bakteriocin stabilan na pH vrednostima između 2 i 9. Dejstvo Tween-a 80 i uree je dovelo do povećane inhibitorne aktivnosti. Prema IEF, bakteriocin G2 je katjonski protein, sa pI vrednošću oko 10, a molekulaska masa određena na osnovu tricin-SDS-PAGE je 2,2 kDa. Probiotski soj L plantarum G2 produkuje antimikrobno jedinjenje proteinske strukture sa karakteristikama bakteriocina. Sinteza bakteriocina je jedna od ključnih osobina probiotskih bakterija koje imaju klinički potencijal kao antiinfektivni agensi, jer značajno povećava verovatnoću njihove in vivo efikasnosti