Detailed state model of CaMKII activation and autophosphorylation

By combining biochemical experiments with computer modelling of biochemical reactions we elucidated some of the currently unresolved aspects of calcium-calmodulin-dependent protein kinase II (CaMKII) activation and autophosphorylation that might be relevant for its physiological function and provided a model that incorporates in detail the mechanism of CaMKII activation and autophosphorylation at T286 that is based on experimentally determined binding constants and phosphorylation rates. To this end, we developed a detailed state model of CaMKII activation and autophosphorylation based on the currently available literature, and constrained it with data from CaMKII autophosphorylation essays. Our model takes exact phosphorylation patterns of CaMKII holoenzymes into account, and is valid at physiologically relevant conditions where the concentrations of calcium and calmodulin are not saturating. Our results strongly suggest that even when bound to less than fully calcium-bound calmodulin, CaMKII is in the active state, and indicate that the autophosphorylation of T286 by an active non-phosphorylated CaMKII subunit is significantly faster than by an autophosphorylated CaMKII subunit. These results imply that CaMKII can be efficiently activated at significantly lower calcium concentrations than previously thought, which may explain how CaMKII gets activated at calcium concentrations existing at synapses in vivo. We also investigated the significance of CaMKII holoenzyme structure on CaMKII autophosphorylation and obtained estimates of previously unknown binding constants

Neurons as a model system for cryo-electron tomography.

Cryo-electron tomography (Cryo-ET) provides unique opportunities to image cellular components at high resolution in their native state and environment. While many different cell types were investigated by cryo-ET, here we review application to neurons. We show that neurons are a versatile system that can be used to investigate general cellular components such as the cytoskeleton and membrane-bound organelles, in addition to neuron-specific processes such as synaptic transmission. Furthermore, the synapse provides a rich environment for the development of cryo-ET image processing tools suitable to elucidate the functional and spatial organization of compositionally and morphologically heterogeneous macromolecular complexes involved in biochemical signaling cascades, within their native, crowded cellular environments

Variability of Morphological Traits of European Beech (Fagus sylvatica L.) Seedlings in Serbia

The results of interpopulation variability of morphometric parameters of European beech (Fagus sylvatica L.) seedlings originating from eight populations from the part of natural distribution area in Serbia are presented in this paper. The studied populations of Dubašnica, Jastrebac, Boranja, Fruška Gora, Mali Pek, Goč, Beljanica, and Javor have various ecological and vegetational characteristics. The results of this paper refer to root collar diameter and height of seedlings at the age of 1+0 and 2+0. Analysis of variance showed a statistically significant difference between the populations in terms of the studied morphological traits of seedlings (p<0.01; α=0.05). Geographical differentiation of the studied populations has not been determined by applying cluster analysis, but the populations are grouped randomly and they indicate the ecotypic nature of beech genetic variation. The results of this research may serve in beech breeding and the available gene pool conservation. Based on the obtained results it can be recommended that in terms of transfer and use of the beech forest reproductive material greater attention should be paid to the ecological conditions of the parent stands and habitats where afforestation has been performed

Variability of Morphological Traits of European Beech (Fagus sylvatica L.) Seedlings in Serbia

The results of interpopulation variability of morphometric parameters of European beech (Fagus sylvatica L.) seedlings originating from eight populations from the part of natural distribution area in Serbia are presented in this paper. The studied populations of Dubašnica, Jastrebac, Boranja, Fruška Gora, Mali Pek, Goč, Beljanica, and Javor have various ecological and vegetational characteristics. The results of this paper refer to root collar diameter and height of seedlings at the age of 1+0 and 2+0. Analysis of variance showed a statistically significant difference between the populations in terms of the studied morphological traits of seedlings (p<0.01; α=0.05). Geographical differentiation of the studied populations has not been determined by applying cluster analysis, but the populations are grouped randomly and they indicate the ecotypic nature of beech genetic variation. The results of this research may serve in beech breeding and the available gene pool conservation. Based on the obtained results it can be recommended that in terms of transfer and use of the beech forest reproductive material greater attention should be paid to the ecological conditions of the parent stands and habitats where afforestation has been performed

Gene pool conservation and tree improvement in Serbia

This paper presents the concepts applied in the gene pool conservation and tree improvement in Serbia. Gene pool conservation of tree species in Serbia includes a series of activities aiming at the sustainability and protection of genetic and species variability. This implies the investigation of genetic resources and their identification through the research of the genetic structure and the breeding system of individual species. Paper also includes the study of intra- and inter-population variability in experiments - provenance tests, progeny tests, half- and full-sib lines, etc. The increased use of the genetic potential in tree improvement in Serbia should be intensified by the following activities: improvement of production of normal forest seed, application of the concept of new selections directed primarily to the improvement of only one character, because in that case the result would be certain, establishment and management of seed orchards as specialized plantations for long-term production of genetically good-quality forest seeds, and the shortening of the improvement process by introducing new techniques and methods (molecular markers, somaclonal variation, genetic engineering, protoplast fusion, micropropagation, etc.)

Detailed state model of CaMKII activation and autophosphorylation

By combining biochemical experiments with computer modelling of biochemical reactions we elucidated some of the currently unresolved aspects of calcium-calmodulin-dependent protein kinase II (CaMKII) activation and autophosphorylation that might be relevant for its physiological function and provided a model that incorporates in detail the mechanism of CaMKII activation and autophosphorylation at T286 that is based on experimentally determined binding constants and phosphorylation rates. To this end, we developed a detailed state model of CaMKII activation and autophosphorylation based on the currently available literature, and constrained it with data from CaMKII autophosphorylation essays. Our model takes exact phosphorylation patterns of CaMKII holoenzymes into account, and is valid at physiologically relevant conditions where the concentrations of calcium and calmodulin are not saturating. Our results strongly suggest that even when bound to less than fully calcium-bound calmodulin, CaMKII is in the active state, and indicate that the autophosphorylation of T286 by an active non-phosphorylated CaMKII subunit is significantly faster than by an autophosphorylated CaMKII subunit. These results imply that CaMKII can be efficiently activated at significantly lower calcium concentrations than previously thought, which may explain how CaMKII gets activated at calcium concentrations existing at synapses in vivo. We also investigated the significance of CaMKII holoenzyme structure on CaMKII autophosphorylation and obtained estimates of previously unknown binding constants

Prostorna varijabilnost morfoloških obilježja iglica populacija jele (Abies alba Mill.) na Balkanskom poluotoku u odnosu na klimatske čimbenike

Interpopulation and intrapopulation variability of three morphological needle traits (length, width and thickness) was investigated in 16 natural silver fir populations in the Balkan Peninsula. The populations represent refugial areas of silver fir (Abies alba Mill.). This paper aims to provide a comprehensive analysis of the influence of climatic factors (mean annual temperature, number of days with temperatures &lt; 0, &gt; 5, &lt; 18, &gt; 18oC, Hargreaves climatic moisture deficit and De Martonne aridity index, on the pattern of morphological needle traits within each population. Populations showed variation in the analyzed morphological needle traits, which could not be clearly defined by any of the analyzed climatic factors. The De Martonne aridity index and Hargreaves climatic moisture deficit had the greatest impact on the trait values, whereas the mean annual precipitation had the lowest. Evolutionary ecology research of the silver fir needle morphology is a valuable contribution to the comprehention of the present genetic variability as a prerequisite for adaptation to the rapid climate change and conservation of the species area in the Balkan Peninsula region.Interpopulacijska i intrapopulacijska varijabilnost triju morfoloških osobina iglica (dužina, širina i debljina) istraživana je u 16 prirodnih populacija jele na Balkanskom poluotoku. Populacije predstavljaju refugijalna područja jele (Abies alba Mill.). Ovaj rad ima za cilj pružiti opsežnu analizu utjecaja klimatskih čimbenika (srednja godišnja temperatura, broj dana s temperaturama &lt; 0, &gt; 5, &lt; 18, &gt; 18°C, Hargreavesov klimatski deficit vlage i De Martonneov indeks aridnosti) na obrazac morfoloških obilježja iglica unutar svake populacije. Populacije su pokazale varijacije u analiziranim morfološkim obilježjima iglica koje se nisu mogle jasno definirati niti jednim od analiziranih klimatskih čimbenika. Najveći utjecaj na vrijednosti obilježja imao je klimatski deficit vlage po Hargreavesu, a zatim najniže vrijednosti srednje godišnje količine oborina. Evolucijska ekološka istraživanja morfologije iglica jele vrijedan su doprinos razumijevanju sadašnje genetske varijabilnosti kao preduvjeta za prilagodbu na brze klimatske promjene i očuvanje vrste na području Balkanskog poluotoka

Variations in fruit traits of wild cherry (Prunus aviumL.) provenances in Serbia

Prirodne populacije divlje trešnje u Srbiji nalaze se blizu južne granice areala ove značajne šumske vrste. Opstanak rubnih provenijencija, u Srbiji i široj regiji jugoistoka Europe, ugrožen je uslijed promjene klime, male veličine populacija, niske kompetitivnosti vrste i dr. Zbog toga se preporučuje umjetno potpomaganje obnove populacija uz proširivanje njihove genske raznolikosti. Iako je poznavanje razine i strukture genetske raznolikosti preduvjet njene učinkovite konzervacije i korištenja, istraživanja ove tematike na divljoj trešnji u regiji su rijetka. Cilj ovog istraživanja bio je utvrditi razinu i obrazac fenotipske varijabilnosti za morfološka svojstva plodova, a raspravljena je i mogućnost povezanosti obrasca fenotipske varijabilnosti s genetskom diferencijacijom provenijencija.Plodovi su prikupljeni u devet prirodnih populacija. Analizirano je deset morfoloških svojstava plodova i utrđena je prosječna klijavost provenijencija. Najmanje varijabilnim pokazalo se svojstvo širina ploda (CV = 6.2%), dok je najvarijabilnije svojstvo bila debljina peteljke (CV = 29.4%). Analizom varijance utvrđeno je da su se provenijencije međusobno statistički značajno razlikovale po svim istraživanim morfološkim svojstvima plodova (p&lt;0.01; &#945; = 0.05), izuzev po svojstvu debljina peteljke (p = 0.92). Iako je varijabilnost između provenijencija bila statistički značajna, razina unutarpopulacijske varijabilnosti bila je znatno veća (41.2-52.1%) od međupopulacijske diferencijacije (5.3-15.2%). Obrazac diferencijacije između provenijencija utvrđen je korelacijskom analizom prosječnih vrijednosti svojstava s klimatsko-geografskim varijablama provenijencija, pri čemu su se debljina ploda i duljina peteljke pokazali kao korisna dijagnostička svojstva. Prosječna debljina ploda bila je signifikantno pozitivno korelirana s nadmorskom visinom (R = 0.69; p = 0.04), prosječnom godišnjom količinom snježne oborine (R = 0.80; p = 0.01) i prosječnim godišnjim brojem stupanj-dana ispod 0°C (R = 0.70; p = 0.04). Prosječna duljina peteljke bila je signifikantno negativno korelirana s prosječnom godišnjom količinom snježne oborine (R = -0.69; p = 0.04), a pozitivno s omjerom godišnje temperature i količine oborina (R = 0.71; p = 0.03). Rezultati su pokazali ekoklinalni obrazac fenotipske diferencijacije provenijencija s obzirom na nadmorsku visinu staništa i s njom koreliranim ekološkim varijablama.Iako su istraživanja obuhvatila fenotipsku varijabilnost plodova iz prirodnih populacija, ovakvi rezultati ukazuju na vjerojatnost genetske diferencijacije provenijencija s obzirom na nadmorsku visinu. To daje temelj preporuci vertikalne sjemenske zonacije areala divlje trešnje u Srbiji, kao i sukladnog korištenja njenog reprodukcijskog materijala u potpomognutoj obnovi. Radi potvrde rezultata ovog istraživanja odnosno preciznije determinacije genetske strukture prirodnih populacija, neophodno je primijeniti metode analiza raznovrsnih fenotipskih svojstava u posebno dizajniranim pokusnim nasadima (npr. testovima provenijencija) kao i analiza prikladnih DNA markera.Natural wild cherry populations in Serbia are a part of the southern border of this valuable forest tree species distribution range. The survival of these marginal provenances in Serbia as well as in the wider region is threatened by climate change, small population sizes, low competitiveness, etc. Therefore, it is recommended to artificially assist population regeneration while increasing their genetic diversity. Although knowledge of the amount and pattern of the specie’s genetic diversity is a prerequisite for its effective conservation and use, related research in the region is scarce. The main goal of this study was to determine the amount and pattern of phenotypic variability of natural wild cherry populations in Serbia. Possible link between revealed pattern of phenotypic variability and genetic differentiation of the provenances was discussed.Fruits were collected in nine natural populations. Ten morphological traits of the fruits were measured, and average germination rate of the provenances was assessed. The least variable trait was the fruit width (CV = 6.2%), while the most variable trait was the petiole thickness (CV = 29.4%). Analysis of variance revealed significant among-provenance variation for investigated fruit characteristics (p &lt;0.01; &#945; = 0.05), except for the petiole thickness (p = 0.92). Although variability among provenances was significant, the level of within-population variability was much higher (41.2-52.1%) than among-provenance differentiation (5.3-15.2%). The pattern of among-provenance variation was determined by a correlation analysis between provenance mean values and their climatic-geographical variables, whereby fruit thickness and petiole length proved to be useful diagnostic traits. Mean fruit thickness was significantly and positively correlated with altitude (R = 0.69; p = 0.04), annual precipitation as snow (R = 0.80; p = 0.01), and annual degree-days below 0 °C (R = 0.70); p = 0.04). The average petiole length was significantly and negatively correlated with the annual precipitation as snow (R = -0.69; p = 0.04), while it was positively correlated with annual heat to moisture index (R = 0.71; p = 0.03). The results revealed an ecoclinal pattern of phenotypic differentiation among the provenances due to their habitat’s altitude and other ecological variables closely related to altitude.Although this study basically dealt with phenotypic variability of wild cherry fruits originating from natural populations, the results indicate likelihood for genetic differentiation of the provenances due to altitude. This likelihood provides the basis for recommending vertical seed zonation, as well as congruent use of reproductive material for assisted restoration of wild cherry populations in Serbia. However, to confirm this presumption of ecoclinal pattern of genetic differentiation, it is necessary to conduct analyses of various phenotypic traits in common garden experiments (e.g. provenance trials) as well as analyses of appropriate DNA markers

Procena genetičke diferencijacije jele (Abies alba Mill.) sa Javora pomoću SSR markera

The process of plant breeding and conservation of gene pool among other things depends on the knowledge of the level of genetic differentiation. The aim of research in this paper was to determine the genetic differentiation of silver fir (Abies albaMill.) populations of regular type and atypical genotypes with pyramidal crown that can be found on Javor mountain, at the site Ogorijevac. The genetic differentiation of silver fir was determined using SSR (Simple Sequence Repeat) markers. Nine SSR pairs of primers gave 29 alleles, while the average number of alleles was 3.2. The primer NFH15 gave the smallest number of alleles (two), while the primer SF78 gave the greatest number of alleles (five). Dice coefficient of the genetic similarity was used to obtain a dendrogram by UPMGA analysis using NTSYSpc statistical program. The genetic similarity recorded among the individuals P1 and P2 was the largest (0.89), while the populations VI and individual P2 showed the lowest similarity (0.61).Based on the cluster analysis it canbe concluded that the studied populations and genotypes of silver fir with different types of crown are clearly differentiated. The basic insight into the level of the genetic diversity of the natural populations of silver fir with the various types of crown has been provided using selected SSR markers. The obtained results can be used for creating further strategy for the conservation of the available gene pool and the regeneration of silver fir forests in Serbia.Proces oplemenjivanja biljaka i konzervacije genofonda između ostalog, zavisi i od poznavanja stepena genetičke diferencijacije. Cilj istraživanja u ovom radu bio je da se utvrdi genetička diferencijacija populacija jele (Abies alba Mill.)normalanog tipa i atipičnih genotipova piramidalanog tipa krošnje, koje se javljaju na planini Javor, na lokalitetu Ogorijevac. Genetička sličnost odnosno različitost jele određena je upotrebom SSR (Simple Sequence Repeat) markera. Devet SSR pari prajmera dalo je ukupno 29 alela, dok je prosečan broj alela bio 3,2. Prajmer NFH15 dao je najmanji broj alela (dva), dok je prajmer SF78 bio sa najvećim brojem alela (pet). Najviša vrednost koeficijenta genetičke sličnosti utvrđena je između genotipova P1 i P2 (0,89), dok je najniža vrednost koeficijenta genetičke sličnosti utvrđena između populacije VI i genotipa P2 (0,61).Koeficijenti genetičke sličnosti po Dice, upotrebljeni su za dobijanje dendrograma pomoću UPMGA analize, koristeći NTSYSpc statistički program. Na osnovu klaster analize može se zaključiti da su istraživane populacije i genotipovi jele sa različitim tipom krošnje jasno izdiferencirani. Upotrebom izabranih SSR markera dat je osnovni uvid u nivo genetičke raznovrsnosti prirodnih populacija jele različitog tipa krošnje. Dobijeni rezultati mogu poslužiti u budućoj strategiji na konzervaciji raspoloživog genofonda i obnavljanju jelovih šuma u Srbiji

Assessment of genetic diversity of silver fir (Abies alba mill.) In Serbia using SSR markers

The paper presents the results of the analysis of genetic variability of eight populations of silver fir (Abies alba Mill.) in Serbia obtained using SSR markers. The genomic DNA was isolated from tissue of needles of all eight populations. Due to the costly and lengthy process a small number of the SSR markers for Abies alba have been developed, so in this study were used the microsatellite markers of related species. The obtained results indicate a low level of the genetic variability between natural populations of silver fir. The total number of alleles detected with nine SSR markers in eight studied populations of silver fir is 28. The range of alleles varies from two for NFF15 to six for SF78 with an average of 3.1 alleles per locus. The mean value of genetic similarity between populations is 0.92. The smallest genetic similarity between pairs of populations is 0.82 (Dubocica Bare and Stara Planina; Dubocica Bare and Tara) and the greatest genetic similarity is 1 (Zlatar and Stara Planina, Zlatar and Tara, Stara Planina and Tara). A basic insight into the level of genetic diversity of natural populations of silver fir in Serbia, which are located in a relatively small area, has been given using a set of SSR markers. The obtained results can be used in the future strategy for the management and regeneration of silver fir forests