54 research outputs found
Pregled najÄeÅ”Äe koriÅ”Äenih 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 oslobaÄanju histamina u nekim stanjima preosetljivosti (alergije tipa I). Karakterizacija alergena podrazumeva odreÄivanje molekulske mase, odreÄivanje strukture, fizikohemijskih svojstava, IgE vezujuÄih osobina i njegovu alergenost. Ovaj rad daje pregled koje se biohemijske i imunohemijske metode najÄeÅ”Äe 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 odreÄena aminokiselinska sekvencija koja bi mogla da predvidi alergenost datog molekula. MeÄutim, informacije dobijene iz detaljnih strukturnih studija ovih molekula Äe doprineti naÅ”em razumevanju patofizioloÅ”kih procesa koji su u osnovi alergijskih oboljenja i unaprediÄe naÄin na koji ih tretiramo
Izolovanje funkcionalne ukupne RNK iz liÅ”Äa 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 liÅ”Äa i polena evropske lipe (Tilia cordata). KoriÅ”Äenjem komercijalno dostupnog pribora za izolovanje RNK iz biljaka izolovana je Äista ukupna RNK iz liÅ”Äa lipe, dok je koriÅ”Äenjem iste metode dobijena degradirana RNK iz polena lipe. UspeÅ”no izolovanje RNK iz liÅ”Äa i polena je dobijeno koriÅ”Äenjem TRIzol reagensa. RNK izolovana ovim metodom je kontaminirana genomskom DNK, koja je uspeÅ”no eliminisana koriÅ”Äenjem enzima DNaze. Dalje su optimizovani i uslovi uklanjanja genomske DNK pomoÄu DNaze. Izolovana ukupna RNK iz oba izvora je dalje uspeÅ”no iskoriÅ”Äena za sintezu prvog i drugog lanca klonske DNK, kao i u reverzno-transkriptivnoj PCR reakciji, dokazujuÄi time da je koriÅ”Äenjem ovog metoda izolovana funkcionalna ukupna RNK. U zakljuÄku, dobijena je Äista i funkcionalna RNK iz liÅ”Äa i polena T. cordata (27,8Ā±7,9 Ī¼g g-1 liÅ”Äa; 25,7Ā±1,1 Ī¼g g-1 polena) koja se može koristiti u daljim molekularno-bioloÅ”kim istraživanjima
Proizvodnja lipaze iz Pseudozyma aphidis i utvrÄivanje aktivnosti i stabilnosti lipaze u polarnim organskim rastvaraÄima
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 utvrÄena je u Å”est razliÄitih medijuma. NajviÅ”a proizvodnja uoÄena je u medijumu gde je glukoza bila izvor ugljenika, a ekstrakt kvasca i natrijum-nitrat izvori azota. PraÄenjem dinamike rasta i proizvodnje lipaze u optimalnom medijumu, uoÄeno je da se najviÅ”a proizvodnja lipaze dostiže pred kraj logaritamske faze rasta, i dostiže vrednost od 35 U cm-3 u petom danu kultivacije, Å”to je Äetri puta veÄa proizvodnja od one do sada prijavljene u literaturi. UtvrÄen je efekat razliÄitih polarnih organskih rastvaraÄa, meÅ”ljivih sa vodom, na aktivnost i stabilnost lipaze iz P. aphidis. HidrolitiÄka aktivnost lipaze prema para-nitrofenil-palmitatu (p-NPP-u) u vo- denoj sredini i organskim rastvaraÄima utvrÄena 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 Äasa inkubacije. Dobijeni rezultati ukazuju da lipaza iz P. aphidis može biti koriÅ”Äena 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 TRIzolā¢ preparation of the total RNA. The total RNA isolated using TRIzolā¢ 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 (27.8Ā±7.9 Ī¼g g-1 leaves; 25.7Ā±1.1 Ī¼g g-1 pollen) could be obtained and was suitable for application in further molecular biology studies
Stabilizacija Ī±-glukozidaze u organskim rastvaraÄima imobilizacijom na makroporoznim (poli) glicidil metakrilatima razliÄitih povrÅ”inskih 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.Ī±-Glukozidaza izolovana iz pekarskog kvasca je imobilizovana na makroporoznim glicidil-metakrilatima razliÄitih povrÅ”inskih karakteristika i veliÄina pora od 44 do 270 nm. Imobilizacija je izvedena glutaraldehidom na polimeru prethodno modifikovanom sa 1,2-diaminoetanom. SpecifiÄna aktivnost dobijenog imobilizovanog enzima je varirala od 27 do 81U/g u zavisnosti od vrste koriÅ”Äenog polimera. Poluživoti imobilizovanog enzima u korastvaraÄima su zavisili od povrÅ”inskih 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. NajveÄa stabilnost u metanolu je dobijena imobilizacijom enzima na polimeru sa veliÄinom pora od 48 nm a u dimetilsulfoksidu na polimeru sa veliÄinom pora od 270 nm
Digestibilnost Ī²-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.Ī²-Laktoglobulin (BLG) je važan nutrijent mleÄnih 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 koriÅ”Äenih za unakrsno povezivanje odreÄena je pomoÄu LC-ESI-MS. Fenolni ekstrakt jabuke sadrži znatne koliÄine glikozida kvercetina, katehine i hlorogensku kiselinu. BLG je unakrsno povezan lakazom u prisustvu fenola jabuke, pri Äemu je polimerizacija uÄinila BLG nerastvornim u reakcionoj smeÅ”i. SDS-PAGE analiza pokazala je da reakciona smeÅ”a sadrži heterogenu smeÅ”u 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 naÄin smanjiti njegovu nutritivnu vrednost. TakoÄe, 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 uÄenja 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 studentsā 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 donoÅ”enje standarda za proces nastave i uÄenja hemije u naÅ”oj zemlji. Standardi za obrazovanje u prirodnim naukama predstavljaju kriterijume za procenu uÄeniÄkih postignuÄa, za procenu da li kvalitet programa i nastave omoguÄava uÄenicima uÄenje prirodnih nauka, za procenu kvaliteta prakse vrednovanja i obrazovne politike. Razvoj standarda u oblasti prirodnih nauka u svetu praÄen je sledeÄim principima: prirodne nauke su za sve uÄenike, uÄenje prirodnih nauka je aktivan proces, prirodne nauke u Å”koli reflektuju intelektualnu i kulturnu tradiciju koja karakteriÅ”e praksu u savremenoj nauci, unapreÄenje obrazovanja u oblasti prirodnih nauka deo je sistematske reforme obrazovanja. TakoÄe, u radu je predložena metodologija za razvoj standarda za proces nastave i uÄenja hemije kod nas.
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
Standardi za proces nastave i uÄenja 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 studentsā 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 donoÅ”enje standarda za proces nastave i uÄenja hemije u naÅ”oj zemlji. Standardi za obrazovanje u prirodnim naukama predstavljaju kriterijume za procenu uÄeniÄkih postignuÄa, za procenu da li kvalitet programa i nastave omoguÄava uÄenicima uÄenje prirodnih nauka, za procenu kvaliteta prakse vrednovanja i obrazovne politike. Razvoj standarda u oblasti prirodnih nauka u svetu praÄen je sledeÄim principima: prirodne nauke su za sve uÄenike, uÄenje prirodnih nauka je aktivan proces, prirodne nauke u Å”koli reflektuju intelektualnu i kulturnu tradiciju koja karakteriÅ”e praksu u savremenoj nauci, unapreÄenje obrazovanja u oblasti prirodnih nauka deo je sistematske reforme obrazovanja. TakoÄe, u radu je predložena metodologija za razvoj standarda za proces nastave i uÄenja 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 kliniÄkog uzorka zdrave osobe. Antimikrobno jedinjenje iz L. plantarum G2, oznaÄeno kao bakteriocin G2, dobijeno iz supernatanta bakterijske kulture, pokazalo je Å”irok spektar antimikrobne aktivnosti, inhibirajiÄi rast svih ispitivanh vrsta roda Lactobacillus, kao i patogenih bakterija Staphylococcus aureus i Salmonella abony. Bakteriocin G2 je osetljiv na proteolitiÄko dejstvo tripsina i proteinaze K. Antimikrobna aktivnost je stabilna u opsegu 40-60Ā°C (15 min), ali se gubi na temperaturi od 80Ā°C. Ustanovljeno je da je bakteriocin stabilan na pH vrednostima izmeÄu 2 i 9. Dejstvo Tween-a 80 i uree je dovelo do poveÄane inhibitorne aktivnosti. Prema IEF, bakteriocin G2 je katjonski protein, sa pI vrednoÅ”Äu oko 10, a molekulaska masa odreÄena 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 kljuÄnih osobina probiotskih bakterija koje imaju kliniÄki potencijal kao antiinfektivni agensi, jer znaÄajno poveÄava verovatnoÄu njihove in vivo efikasnosti
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