Genetic transformation of Centaury (Centaurium erythraea Rafn.) using AtCKX1 and AtCKX2 genes

Biljna vrsta Centaurium erythraea Rafn., u našem narodu poznata kao kičica, od davnina se upotrebljava kao lek za sniženje temperature i nivoa šećera u krvi, lečenje anemije, žutice i gihta, za jačanje apetita i povećanje stomačne sekrecije. Zbog poznatih lekovitih svojstava, kičica se nalazi u mnogim farmakopejama. Kao početni materijal za uspostavljanje kulture korenova in vitro korišćeni su vrhovi adventivnih korenova prosečne dužine ~10 mm koji su izolovani od sterilno dobijenih klijanaca kičice i dalje su gajeni na čvrstoj MS hranljivoj podlozi bez regulatora rastenja. Proces organogeneze u kulturi korenova kičice je spontan. Posle sedam dana gajenja u kulturi na bazalnom delu početnog eksplantata uočavaju se prve promene. Koren zadebljava i tkivo počinje da puca celom dužinom bazalnog dela. Tokom četvoronedeljnog pasaža, duž razraslog dela eksplantata, formira se veliki broj adventivnih pupoljaka. U cilju dobijanja transgenih biljaka sa sniženim nivoom endogenih citokinina, za transformaciju su korišćena dva bakterijska soja Agrobacterium tumefaciens GV3101 koji nose pBinHTX plazmid sa dva različita gena, AtCKX1 odnosno AtCKX2, koji kodiraju enzim citokinin oksidazu/dehidrogenazu (CKX). Efikasna integracija AtCKX1 gena (30% ) i AtCKX2 gena (28.2% ) određena je PCR analizom genomske DNK. U cilju što boljeg određivanja nivoa ekspresije AtCKX gena kičice prvo je bilo neophodno urađena je optimizacija PCR protokola sa prajmerima za qPCR. Ovaj postupak sastojao se u pronalaženju optimalne temperature vezivanja prajmera uz istovremenu primenu različitih aditiva odnosno pojačivača PCR reakcije. Dodavanje 5% DMSO-a u qPCR reakcije značajno je poboljšalo specifičnost amplifikacije AtCKX1 i AtCKX2 gena kičice. qRT-PCR analizom potvrđena je ekspresija AtCKX1 i AtCKX2 gena kod 50% odnosno 64% analiziranih linija. U ovom radu uspešno je pokazano da ekspresija AtCKX gena u transgenim biljkama kičice značajno utiče na metabolizam citokinina. Ključni faktor u kontroli nivoa citokinina u biljnim tkivima predstavlja CKX enzim, jedini do sada poznati enzim koji učestvuje u katabolizmu citokinina. Specifična aktivnost CKX enzima određivana je u izdancima i korenovima svih transgenih AtCKX linija. Analiza količine i sadržaja ukupnih kao i pojedinačnih grupa endogenih citokinina netransfromisanih biljaka kičice omogućila je, po prvi put, određivanje citokiniskog profila kičice in vitro...Plant species Centaurium erythraea Rafn., commonly known as common centaury, has been used for centuries to cure febrile conditions, regulate blood sugar, treat anemia, jaundice and gout and to increase appetite and stimulate digestion. Because of its numerous therapeutic properties centaurii herba is officially recognized as a drug in a number of pharmacopoeias. Root tips of .10 mm average length, isolated from seedlings obtained under sterile conditions, were used as initial explants in this work. The in vitro culture of non-transformed centaury roots was established on solid half-strength MS nutrient medium without growth regulators. Organogenesis is a spontaneous process in centaury root culture. The first changes were noticeable on the basal part of the initial explants, after seven days of growth on MS medium. The root explants thickened and began to burst over the entire length of the basal part. During four weeks of subculture, a large number of adventitious buds were observed on initial explants. In order to obtain transgenic centaury plants with lowered endogenous cytokinin levels, in vitro cultivated root explants were transformed with Agrobacterium tumefaciens GV3101 bearing a pBinHTX plasmid with either AtCKX1 or AtCKX2 genes encoding isoforms of cytokinin oxidase/dehydrogenase (CKX). PCR analysis of genomic DNA confirmed the effecient integration of both AtCKX1 (30%) and AtCKX2 (28.2%) genes. In order to investigate the expression of AtCKX genes in transgenic centaury plants, an optimization of PCR protocol with qPCR primers was necessary. Optimization of PCR protocol implicated finding the optimal annealing temperature and simultaneous application of different PCR additives and enhancers. Addition of 5% DMSO significantly improved the specificity of qPCR amplification of AtCKX1 and AtCKX2 genes. qRT-PCR analysis indicated that AtCKX1 and AtCKX2 genes were expressed in 50% and 64% of the analyzed transgenic lines, respectively. In this work we clearly demonstrate a significant effect of AtCKX genes on cytokinin metabolism in transgenic centaury plants. Since CKX is the only known enzyme involved in cytokinin catabolism, it is considered a key factor in controlling the cytokinin levels in plant tissues. CKX enzyme activity was determined in shoots and in roots of all AtCKX transgenic lines. The analysis of the content of total cytokinins and individual groups of endogenous cytokinins in non-transformed centaury plants enabled, for the first time, a determination of cytokinin profile of this species in vitro..

Pseudotumorous silicosis in a founder

Autori su prikazali slučaj pseudotumorozne forme silikoze kod livca starog 76 godina. Tumori su lokalizovani obostrano infraklavikularno, a desno je tumor u raspadu. U levom hilusu je kalcifikovana uvećana limfna žlezda u vidu »Eierschalenverkalkung«. I pored masivnih tumora bolesnik je imao relativno sačuvanu funkciju pluća, jer su prevalirale restriktivne promene u plućima bez značajnije opstruktivne komponente, čime se tumači i odsustvo znatnijih poremećaja kardiovaskularnog aparata.A case of pseudotumorous silicosis in a founder aged 76 is described. Tumors were localized bilaterally infraclavicularly, the right tumor was in the state of decomposing. In the left hilus were found the enlarged egg-shell like calcifications of the lymph gland. Inspite of massive tumors the lung function of the patient was relatively preserved owing to the prevalence of restrictive pulmonary changes without a significant obstructive component, what also accounts for the absence of severe cardiovascular disorders

The Impact of One Heat Treated Contact Element on the Coefficient of Static Friction

The subject of the paper includes theoretical considerations, the conducting of experimental tests, and the analysis of exposed test results related to determination of the coefficient of static friction of previously heat-treated contact pairs. One contact element is previously, before the procedure of determining the coefficient of static friction, heated at temperatures in the range of ambient temperature to 280°C and then cooled down to ambient temperature. The results of experimental tests of five different materials show that depending on the heat treatment of one contact element, there is a significant decrease in the coefficient of static friction. The authors of the paper consider that the reasons for the decreasing coefficient of static friction are related to oxide formation and changes in the surface layer of the contact element which is previously heat-treated

Die Veranderungen des Schneidezahnuberbisses bei Kindern in Zahnwechsel

Autori su ispitivali individualne promene dubine preklopa sekutića, kod dece sa neutrookluzijom, u periodu od 7. do 10. god, i od 10. do 13. god. Promene dubine preklopa sekutića u obe grupe ispitanika, 257 u grupi od 7. god. i 333 u grupi od 10 god, podeljene su u podgrupe: otvoren, ivični normalan i dubok preklop sekutića. Prosečna dubina preklopa sekutića, kod dece od 7. do 10. god, povećava se za 0,75 mm, a od 10. do 13. god, za 0,30 mm. Kod grupa sa očuvanim prvim stalnim molarima, ovo povećanje je nešto manje izraženo. Individualnim posmatranjem svakog ispitanika sa otvorenim i ivičnim preklopom sekutića, nalazimo spontanu korekciju kod skoro 75% dece, u periodu od 7. do 10. god, dok kod grupe dece sa očuvanim prvim stalnim molarima, tek u 50% slučajeva. Kod dubokog preklopa sekutića, u istom periodu, pogoršanje je uočeno kod 57,90% dece. Kod dece drugog perioda ispitivanja (10— 13) god, promene ivičnog preklopa, u smislu spontane korekcije, manje su, ali je > i manji procent slučajeva povećanja preklopa. Ekstrakcije prvog stalnog molara imaju veći uticaj kod dece sa otvorenim zagrižajem, nego kod slučajeva sa izraženim dubokim preklopom sekutića.The individual changes in the depth of the incisor overlap were analysed in children with neutroocclusion aged from 7— 10 and from 10— 13 years. The variations of the depth of the incisor overlap in both groups including 257 subjects aged 7 years and 333 subjects aged 10 years have been divided into the following subgroups: open overlap, marginal normal and deep overlap of the incisors. The average depth of the incisor overlap in children aged 7— 10 increases for 0.75 mm and in those aged 10— 13 for 0.30 mm. In groups with preserved firs t permanent molars this increase is less expressed. The individual observation of each subject with open and marginal overlap of the incisors shows spontaneous corrections in over 75% of children aged 7— 10 years and only in 50% of children with preserved firs t permanent molars. In cases with deep incisor overlap an aggravation is found in 57.90% of childeren. In children aged 10— 13 the changes of the marginal overlap in the sense of spontaneous correction are less expressed as well as the percentage of cases with an increased overlap. The extraction of the first permanent molar have higher effect in children with open overlap than in cases with expressed deep overlap of the incisors.Die Autoren haben die individuellen Veränderungen des Schneidezahnüberbisses bei Kindern mit Neutrookklusion im Alter von 7 bis 10 Jahren und im Alter von 10 bis 13 Jahren, geprüft. Die beiden Probanden-Gruppen 257 im Alter von 7 bis 10 Jahren, und 333 im Alter von 10 Jahren — wurden in folgende Untergruppen eingeteilt: offener Biss, Normalbiss und tiefer Schneidezahnüberbiss. Die durchschnittliche Tiefe des Schneidezahnüberbisses vergrössert sich bei Kindern von 7 bis 10 jahren um 0,75 mm, im Alter von 10 bis 13 Jahren um 0,30 mm. Bei Kindern m it erhaltenen bleibemden ersten Molaren is t diese Zunahme etwas geringer ausgedrückt. Bei der individuellen Beobachtung jedes Probanden mit offenem Biss und Kantenüberbiss, fanden w ir eine spontane Korrektur bei fast 75% der Kinder im Alter von 7 bis 10 Jahren, während bei Kindern mit erhaltenen ersten bleibenden Molaren der Perzentsatz bloss 50% betrug. Bei tiefem Überbiss ist in der gleichen Altersperiode eine Verschlimmerung bei 57,90% der Kinder festgestellt worden. Bei der zweiten Probandengmppe (10 bis 13 Jahre), sind Veränderunge des Kantenüberbisses im Sinne einer Spontankorrektur geringer, aber auch Fälle m it Vergrösserung des Überbisses sind seltener. Die Extraktion des ersten bleibenden Molaren ist schwerwiegender bei offenem Biss als bei ausgeprägtem Schneidezahnüberbiss

Ethylenediammonium aquabis(malonato)oxovanadate(IV)

In the anion of the title compound, (C2H10N2)[VO(H2O)(C3H2O4)(2)] or H(2)en[VO(mal)(2)H2O], vanadium(IV) is distorted-octahedrally coordinated by six donor O atoms. The two malonate ligands are situated in the equatorial plane, whereas the oxo and the water ligand occupy axial positions. The apical V=O bond exhibits a strong trans influence. The anion possesses crystallographically imposed C-2 symmetry, with the central V atom, the oxo and the water ligand lying on the twofold axis

Volatile Constituents of Different Plant Parts and Populations of Malabaila aurea Boiss. from Montenegro

The volatile constituents of different plant parts and populations of Malabaila aurea Boiss. from Montenegro were obtained by simultaneous distillation-extraction and analyzed by GC-FID and GC-MS. A total of 12 samples were examined and 45 compounds were identified. The volatile content of different M. aurea populations was very similar, while the volatile fractions obtained from different plant parts showed significant qualitative and quantitative differences. The most abundant compounds found in stems & leaves were apiole (51.0-56.3%), myristicin (16.3-25.4%), and falcarinol (4.1-10.7%). The roots showed the same major components, but with different relative abundances: 30.9-49.1% of apiole, 12.9-34.7% of falcarinol, and 9.9-31.1% of myristicin. The volatile constituents of fruits & flowers were remarkably different, containing up to 71.2-80.5% octyl butyrate, 11.4-18.0% octanol, and 2.7-6.8% octyl hexanoate. The results were discussed as possible indication of relatedness of Malabaila aurea and Pastinaca sativa (parsnip)

Uticaj nivoa mlečnosti na osobine plodnosti krava crno bele rase

In this study a relationship between the milk yield and following reproduction traits: number of inseminations per conception [AIS], service period [SP], age at calving [AC], calving interval [CI] and birth weight [BW] is studied. The research involved dairy cows of European type of Black and White cattle which are being intensively improved by the genes of Holstein - Friesian breed. Mean values of general average for number of inseminations per conception, service period, age at calving, calving interval and birth weight are: 2.00; 98.78; 1088.08; 376.70 and 36.40, respectively. In general, the estimated phenotypic correlations (rp) between the milk yield over standard lactations and fertility traits were positive and unfavorable, and ranged from 0.24 for the relationship between milk yield and birth weight to 0.70 for relationship between milk yield and age at calving.U radu je ispitivan odnos između prinosa mleka i sledećih osobina plodnosti: indeks osemenjavanja [IO], servis period [SP], uzrast pri teljenju [UT], međutelidbeni interval [MI] i masa teladi pri rođenju [MTR]. Ispitivanja su se odnosila na mlečne krave evropskog tipa crno-belih goveda koje se intenzivno oplemenjuju genima holštajn-frizijske rase. Za analizu je korišćen Metod najmanjih kvadrata - LSMLMW (HARVEY, 1990), fiksni model. Srednje vrednosti opšteg proseka za indeks osemenjavanja, servis period, uzrast pri teljenju, međutelidbeni interval i masu teladi pri rođenju su: 2,00; 98,78; 1088,08 ; 376,70 i 36,40, respektivno. Procenjene fenotipske korelacije (rp) između prinosa mleka i osobina plodnosti generalno su bile pozitivne i nepovoljne, i imale su vrednosti od 0,24 za odnos između prinosa mleka i masu teladi pri rođenju do 0,70 za odnos između prinosa mleka i uzrasta pri teljenju

Torrential floods and town and country planning in Serbia

Torrential floods are the most frequent natural catastrophic events in Serbia, causing the loss of human lives and huge material damage, both in urban and rural areas. The analysis of the intra-annual distribution of maximal discharges aided in noticing that torrential floods have a seasonal character. The erosion and torrent control works (ETCWs) in Serbia began at the end of the 19th century. Effective protection from torrential floods encompasses biotechnical works on the slopes in the watershed and technical works on the torrent beds, within a precisely defined administrative and spatial framework in order to achieve maximal safety for people and their property. Cooperation to overcome the conflicts between the sectors of the water resources management, forestry, agriculture, energetics, environmental protection and local economic development groups is indispensable at the following levels: policy, spatial planning, practice, investments and education. The lowest and most effective level is through the Plans for Announcement of Erosive Regions (PAERs) and the Plans for Protection from Torrential Floods (PPTFs), with Hazard Zones (HZs) and Threatened Areas (TAs) mapping on the basis of the hydrologic, hydraulic and spatial analysis of the factors that are important for the formation of torrential floods. Solutions defined through PAERs and PPTFs have to be integrated into Spatial Plans at local and regional levels

Sinteza i karakterizacija nikal(II) i bakar(II) kompleksa sa polidentatnim dialkil ditiokarbaminskim ligandom 3-ditiokarboksi-3-aza-5-aminopentanoatom

Square planar complexes of Ni(II) and Cu(II) with potassium 3-dithiocarboxy-3-aza-5-aminopentanoate have been prepared by direct synthesis. The obtained neutral complexes were characterized by elemental analysis, magnetic susceptibility measurements, infrared and electronic spectra. The thermal behaviour of both the Ni(II) and Cu(II) complexes, and the ligand itself was investigated by DSC and TG.Direktnom sintezom između nikal(II)- i bakar(II)- soli i kalijum 3-ditiokarboksi-3-aza-5-aminopentanoata nagrađeni su odgovarajući kompleksi kvadratno-planarne strukture. Izolovani neutralni kompleksi karakterisani su elementalnom analizom, infracrvenom i elektronskom apsorpcionom spektroskopijom, kao i merenjem magnetnih susceptibiliteta. Termičko ponašanje kompleksa i liganda ispitivano je primenom DSC i TG metoda