La spectrométrie de masse : un couteau suisse pour disséquer la structure et la fonction du spermatoprotéasome

Le protéasome est le complexe enzymatique protéolytique principal de la cellule. Son cœur catalytique (20S) est formé de quatre anneaux heptamériques. Son activité et sa spécificité de substrat peuvent être régulées par les complexes 19S, PA28alphaß, PA28gamma et PA200 ainsi que par des sous-unités 20S alternatives. La spermatogenèse est un processus de différenciation des cellules germinales mâles: les spermatogonies (SPG) se transforment en spermatocytes (SPC), en spermatides (SPT) puis en spermatozoïdes. Ce processus requière une protéolyse intense. Le spermatoprotéasome (spt20S) est spécifique des gamètes en développement et essentiel à la spermatogenèse. Il diffère du protéasome standard (std20S) par la sous-unité alpha4s qui remplace la sous-unité constitutive alpha4. Le spt20S joue un rôle important avec PA200 dans la progression de la méiose, mais les mécanismes qui le rendent différent du std20S restent inconnus. Nous avons établi des stratégies protéomiques complémentaires pour caractériser les complexes du protéasome immunopurifiés à partir de testicules. L'analyse Top-Down de protéasome purifié, nous a permis de montrer pour la première fois qu'alpha4s et alpha4 portent les mêmes MPTs. La protéomique Bottom-Up, nous a permis de comparer les immunopurifications (IPs) de protéasomes totaux avec celles obtenues avec un anticorps spécifique du stp20S que nous avons développé. Nous avons établi qu'alpha4 et alpha4s ne coexistent pas dans le même 20S, bien qu'ils soient presque également abondants dans les testicules. Nous avons également trouvé plus de 19S et de PA200 liés au spt20S qu'au std20S. Les autres protéines préférentiellement associées au spt20S incluent PI31 et Fbxo7 qui sont cruciales pour la fertilité et d'importants médiateurs du transport du protéasome et de l'ancrage des E3 ligases - deux processus qui semblent cruciaux pour la fonction du spt20S pendant la spermatogenèse. Nous avons ensuite obtenu des cellules germinales à différents stades de la différenciation et l'analyse protéomique des lysats ainsi que des IPs, nous a permis d'établir un interactome dynamique du protéasome tout au long de la spermatogenèse. Nous avons observé un changement total du std20S au spt20S entre les SPG pré-méiotiques et les SPC/SPT méiotiques et post-méiotiques. Un changement d'expression semble responsable, plutôt qu'une incorporation préférentielle d'alpha4s. En entrant dans la méiose, l'association de PA200 avec le protéasome a augmenté 7 fois, confirmant son importance dans le développement des gamètes. Bien que PA200 soit d'après la littérature le principal activateur du spt20S, nous montrons que le 19S est est en réalité majoritaire, lié à 60% des 20S dans les SPC - une activation du protéasome sans précédent. De nombreux partenaires du spt20S sont identifiés à la fois dans les cellules germinales et dans les testicules entiers, montrant la robustesse de nos méthodes. Ceux-ci incluent des protéines synaptonémales, de nombreuses protéines impliquées dans l'ubiquitylation, le cycle cellulaire et la progression méiotique ainsi que le transport cellulaire, en accord avec les fonctions du spt20S proposées dans la littérature. Le passage d'alpha4 à alpha4s semble crucial pour la méiose, mais quelles sont les bases moléculaires de cette transition ? L'échange hydrogène-deutérium nous a permis de montrer pour la première fois que les deux protéasomes présentent des interfaces d'interaction différentes : alpha4s contient des régions plus flexibles qu'alpha4. Cette découverte est confirmée par des pull-down in vitro montrant que le 19S se lie plus fortement au spt20S qu'au std20S, expliquant la hausse d'activité protéolytique pendant la méiose. L'activité trypsique du spt20S est plus élevée que celle du std20S in vitro, ce qui pourrait refléter la nécessité de dégradation des histones. Globalement, nos données révèlent un processus de régulation du spt20S qui est plus complexe que ce qui avait été suggéré précédemment et jettent les bases des différences structurales et fonctionnelles entre le spt20S et le std20S.The proteasome is the main enzymatic complex for targeted proteolysis in the cell. Its core complex (20S) consists of four stacked heptameric rings and requires activator complexes: 19S, PA28alphaß, PA28gamma and PA200, which regulate 20S activity and substrate specificity. Alternative 20S subunits exist to further modulate the proteasome activity. Spermatogenesis is a process of male germ cell differentiation, where spermatogonia (SPG) transform through spermatocyte (SPC), then spermatid (SPT) stages, to become spermatozoa. This process requires intense proteolysis. The spermatoproteasome (spt20S) is specific to the developing gametes and essential for spermatogenesis. It differs from the standard proteasome (std20S) by only one subunit - alpha4s, which replaces the constitutive alpha4 subunit. Together with PA200, the spt20S plays an important role in meiosis progression, however, the mechanisms that make it different compared to std20S remain unknown. We established complementary proteomic pipelines for characterisation of proteasome complexes in the testes, combining immunopurification (IP) and mass spectrometry (MS) analysis. Our Top-Down analysis of purified proteasome, showed for the first time that both alpha4 and alpha4s carry the same PTMs. Using Bottom-Up proteomics we compared the interactome of total proteasomes with that of the spt20S only, obtained using a specific antibody we developed for this purpose. We established that alpha4 and alpha4s do not co-exist in the same 20S, although they are almost equally abundant in the testes.  We also measured that 19S and PA200 regulators were bound in higher ratios to the spt20S compared to std20S. Among other preferentially-associated proteins of spt20S were PI31 and Fbxo7, both shown to be crucial for fertility. They mediate proteasome transport and docking of E3 ligases - both processes that could be crucial for spt20S function. We then obtained germ cells at different differentiation stages and performed a proteomics analysis of both lysates and IP-ed proteasome complexes, to establish a dynamic image of the proteasome throughout spermatogenesis. We observed a complete shift from std20S to spt20S between pre-meiotic SPG and meiotic and post-meiotic SPC and SPT cells. We explained this by a shift in expression, rather than preferential incorporation of alpha4s. Upon entering meiosis, the PA200 association with core proteasome increased 7-fold, marking its importance in gamete development. Although PA200 was represented in literature as the main spt20S interactor, we show that 19S was undoubtedly stoichiometrically dominant, occupying 60% of the existing 20S in SPCs - an unprecedented proteasome activation. Identified spt20S-interacting proteins largely correlated with previous interactome analysis on the whole testes, showing robustness of our methods. We identified synaptonemal proteins bound exclusively to spt20S and numerous proteins involved in ubiquitylation, cell cycle and meiotic progression as well as cellular transport, which fits the current model of spt20S role, proposed by earlier work. The shift from alpha4 to alpha4s in meiosis was shown to be crucial, but what is the molecular basis for this transition? The hydrogen-deuterium exchange experiment coupled to MS helped us to show for the first time that the two proteasomes exhibit different binding interfaces: alpha4s contains regions that are more flexible compared to alpha4. We further supported this finding with pull-down assays, which showed that 19S binds more strongly to the spt20S than to std20S, which would explain the increase in proteolytic activity required during meiosis. The spt20S showed a higher tryptic activity compared to the std20S in vitro, which might reflect a particular need for histone degradation. Altogether, our data reveal a more complex process of spt20S regulation than previously suggested and set the basis for structural and functional differences between the spt20S and std20S

THE APPLICATION OF INTERVAL CALCULUS TO ESTIMATION OF PLATE DEFLECTION BY SOLVING POISSON’S PARTIAL DIFFERENTIAL EQUATION

This paper describes the application of interval calculus to calculation of plate deflection, taking in account inevitable and acceptable tolerance of input data (input parameters). The simply supported reinforced concrete plate was taken as an example. The plate was loaded by uniformly distributed loads. Several parameters that influence the plate deflection are given as certain closed intervals. Accordingly, the results are obtained as intervals so it was possible to follow the direct influence of a change of one or more input parameters on output (in our example, deflection) values by using one model and one computing procedure. The described procedure could be applied to any FEM calculation in order to keep calculation tolerances, ISO-tolerances, and production tolerances in close limits (admissible limits). The Wolfram Mathematica has been used as tool for interval calculation

Uticaj načina dimljenja na prinos, hemijski sastav i senzorna svojstva dimljene ribe

This study compared the effects of smoking processes on the production weight losses, chemical and sensory characteristics of smoked fish - rainbow trout (Oncorchynchus mykiss, Walbaum) and common carp (Cyprinus carpio L). The averge production weight losses, determined in the conditions of our experiments, ranged from 9.60 % (cold smoked carp fillets) to 20.90% (cold smoked trout fillets). The smoking process reduced the moisture content and increased the protein, lipids and NaCl content in all investigated samples. The overall sensory quality of coldsmoked trout fillets was best estimated (4.66), while the overall sensory quality of coldsmoked carp fillets was the least acceptable (3.94)

Postupci pripreme ostataka rezidbe iz višegodišnjih zasada

Previous investigations have proved the fact that Serbia has a substantial renewable energy potential amounting to more than 3 million tons petrol equivalents annually. The share of biomass accounts for approx. 80% of the potential. At the same time, total consumption of fossil fuel is 12 million tons petrol equivalents. If Serbia, spending about 2.5 million tons petrol equivalents per year, were to use only 10% of its biomass potential, i.e. 2.6 million tons petrol equivalents in order to meet household heat energy needs, about 60 million Euros would be saved as a result of import decrease. Optimal technological and technical solutions should be defined with regard to collecting, loading, transporting and preparing pruning remains in orchards and vineyards with the aim of obtaining energy. This is expected to have a significant influence on the energetic efficiency of fruit growing and is an issue of major concern. In our country collecting, preparing and using pruning remains is not widely used due to the irrational disposal of energy and extensive production. Pruning remains have their advantage as sustainable energy source being at the site of consumption or its vicinity. The simplest and oldest way of using pruning remains as energents in the process of burning and producing heat energy is to burn the unchanged remains. Considering the bulk of it which is characterized by the irrational transport, impeded loading, unloading, storing and use in combustion equipments.Sva dosadašnja istraživanja potvrđuju činjenice da u Srbiji postoji značajan energetski potencijal obnovljivih izvora energije u iznosu od više od tri miliona tona ekvivalentne nafte godišnje. U tom potencijalu sa oko 80% ima učešće biomasa. Istovremeno, ukupna potrošnja fosilnih goriva je na nivou od 12 miliona tona ekvivalentne nafte. Kada bi se iskoristilo samo deset odsto potencijala biomase u iznosu od 2,6 miliona tona ekvivalentne nafte radi obezbeđenja toplotnih energetskih usluga u sektoru domaćinstava, za šta se godišnje u Srbiji potroši oko 2,5 miliona tona ekvivalentne nafte, ušteda na račun smanjenog uvoza iznosila bi oko 60 miliona evra godišnje. Projektovnje optimalnih tehnologija i tehničkih rešenja prikupljanja, utovara, transporta i neposredne pripreme ostataka rezidbe voćaka i vinove loze za dobijanje energije, presudno utiče na energetsku efikasnost voćarske proizvodnje i predstavlja veoma aktuelni problem. Zbog neracionalnog raspolaganja energijom i ekstenzivnije proizvodnje, u našim uslovima, prikupljanje, obrada, priprema i korišćenje biljnih ostataka nisu našli širu primenu. Biomasa u vidu ostataka rezidbe kao obnovljivi izvor energije ima i prednost u tome što se najčešće nalaze na mestu potrošnje ili u njihovoj blizini. Najjednostavniji i najstariji način korišćenja ostataka rezidbe kao energenta u procesima sagorevanja i dobijanja toplote je kada se u neizmenjenom obliku obavi proces sagorevanja. Zbog kabaste forme koju karakteriše mala zapreminska masa, uslovljava veoma malu racionalnost u transportu kao i otežan utovar, istovar, skladištenje i samu upotrebu u gorionicima

Stanje i mogućnosti korišćenja biomase kao obnovljivog izvora energije

Biomass is made of plant material used as fuel or transformed into other forms prior to combustion. Biomass is biodecomposable fraction of products: agricultural waste and remains (including plant and animal material), forstery and wood industry but also biodecomposable fractions of industrial and municipal solid waste. The use of biomass as energy source dates far back into the past. In most underdeveloped countries it still represents the major source of energy in households. However, currently the use of biomass as renewable energy source apart from household continues to rise. Of the renewable sources of energy, the biomass potential ranks first. The bioenergy potential is huge and widespread. Currently biomass is the major source of total world energetic needs. Of the available renewable energy sources it accounts for 12% (50 EJ/year) of the total world needs (406 EJ/year). The use of biomass is primarily based on agricultural and forestry waste. Due to its bioenergetic properties it can be used in small, mid and large plants for numerous processing and utilization schemes.Biomasa se definiše kao biljni materijal, koji se koristi direktno kao gorivo, ili pretvara u druge oblike pre sagorevanja. Biomasa je biorazgradljiva frakcija proizvoda: otpaci i ostaci iz poljoprivrede (uključujući biljne i životinjske supstance), šumarstva i prateće industrije, kao i biorazgradljive frakcije industrijskog i gradskog otpada. Korišćenje biomase kao izvora energije datira od davnina, tako da u mnogim nerazvijenim zemljama sveta i dalje predstavlja osnovno gorivo u domaćinstvima. S obzirom da poslednjih godina se počinje sa organizovanim korišćenje biomase izvan domaćinstava i da se računa u značajan energetski resurs, tretira se kao novi obnovljiv izvor energije. Od obnovljivih izvora energije energetski potencijal biomase je na prvom mestu. Potencijal za bioenergiju je veoma velik i vrlo rasprostranjen širom sveta. Danas je biomasa već glavni izvor ukupnih svetskih energetskih potreba, od svih raspoloživih obnovljivih izvora energije, i dostiže 12% (50 EJ/god.) od ukupnih svetskih potreba (406 EJ/god.). Korišćenje biomase je uglavnom bazirano na ostacima poljoprivrede i šumarstva. Zbog osobine bioenergije da se može koristiti u malim, srednjim i velikim postrojenjima primenjena je u velikom broju procesnih i korisničkih šema

Izbor TMA za duboku obradu zemljišta pri podizanju višegodišnjih zasada

The choice of the appropriate aggregate for deep tillage is an issue of major concern in the technology of orchard establishment. Conventional technologies include trenching or subsoiling used to improve the mechanical properties of the soil intended for orchard establishment. The objective of the study was to analyze deep tillage from the technological standpoint prior to orchard establishment. In addition, the aim was to give an overview of the equipment and machinery used in the trenching stage. The analysis included the results of friction in the trenching phase on heavy soils considering labor input, depth and speed. The results were used to analyze input during trenching. A comparative analysis was conducted regarding energy input in tenching and subsoiling. The conclusion which tends to emerge is the enormous amount of energy needed in both trenching and subsoiling. Energy requirements in the trenching stage were 2.5-fold greater in relation to subsoiling. If possible, trenching should be substituted by subsoiling due to a lower enegy input not excluding other advantages.Jedan od značajnih problema u tehnologiji podizanja višegodišnjih zasada, predstavlja adekvatan izbor TMA za duboku obradu. Primenom konvencionalnih tehnologija obrade ova agrotehnička mera se ostvaruje rigolovanjem ili podrivanjem a ima za cilj poboljšanje mehaničkih osobina zemljišta namenjenog gajenju višegodišnjih zasada. U radu je obrađen tehnološki aspekt duboke obrade koja se obavlja pre podizanja višegodišnjih zasada. Pored toga dat je opis pojedinih oruđa i mašina koje se primenjuju u rigolovanju i podrivanju. Analizirani su rezultati otpora pri rigolovanju na vrlo teškim zemljištima u funkciji radnog zahvata, dubine rada i brzine kretanja. Na osnovu vrednosti otpora analiziran je učinak pri rigolovanju. Zatim, obavljena je komparativna analiza rezultata utroška energije kod rigolovanja i podrivanja. Zaključak je da rigolovanje i podrivanje angažuju veliku količinu energije. Za približne uslove, rigolovanje u odnosu na podrivanje do 2,5 puta više angažuje energije. Treba težiti, gde agrotehnički zahtevi omogućuju, da se rigolovanje zameni podrivanjem zbog manjeg utroška energije i drugih prednosti

Eksploatacioni i energetski aspekti pripreme zemljišta za podizanje višegodišnjih zasada

Intensive soil preparation including both mechanical and human labour is required prior to the planting and establishment of orchards. Due to energy deficiency during the past years attention was focused on rational technology with the aim of decreasing energy input in the cultivation stage. Trenching up to 40 cm (in some cases up to 120 cm) is considered the most demanding operation in the soil cultivation stage. Attempts were made to substitute the trenching operation, however it is still the most important stage on some soil types in practice. In our practice tenching is the traditional technology of soil cultivation for orchard establishment. It is of major importance for loosening the B glay horizont in heavy soil types. It also contributes to favourable conditions for the development of the root system. The aim was to analyze the energetic aspect of trenching by determining fuel consumption per unit of time and cultivated area prior to orchard establishment. In addition, the structure of working hours was monitored as well. Also, technical features of the trenching aggregate were given. The conclusion emerges that energy requirements in the trenching stage may amount to 250 kWh/ha at 72 cm cultivation depths. Therefore, attention should be focused on rational soil cultivation prior to orchard establishment.Zasnivanje višegodišnjih zasada zahteva intenzivnu pripremu zemljišta pre sadnje što predstavlja veliko angažovanje mehaničkog a i ljudskog rada. Obzirom da se poslednjih godina zbog deficita energije, velika pažnja poklanja primeni racionalnih tehnologija kojima će se postići smanjenje utroška energije pri obavljanju operacija u obradi. Kao najzahtevnija operacija u pripremi zemljišta predstavlja duboka obradarigolovanje zemljišta na dubini preko 40 cm, a za neke zasade čak i do 120 cm. I pored pokušaja supstitucije ove operaciji u pripremi zemljišta u praksi još uvek ima značajnu primenu što predstavlja obaveznu meru na određenim tipovima zemljišta Činjenica je da u našoj praksi još uvek preovlađuju tradicionalne tehnologije obrade koje uključuju primenu rigolovanja kao operacije u pripremi zemljišta pre podizanja višegodišnjih zasada. Značaj ovakve duboke obrade ogleda se u rastresanju B glejnog horizonta kod teških tipova zemljišta. Zatim, obezbeđenje povoljnih uslova za razvoj korenovog sistema, pogotovu ako se ima u vidu da višegodišnji zasadi ostaju i nekoliko desetina godina na jednom mestu. U radu je obrađen energetski aspekt duboke obrade rigolovanjem utvrđivanjem potrošnje goriva po jedinici vremena i obrađenoj površini koja se obavlja pre podizanja višegodišnjih zasada. Ispitivanjima su bili obuhvaćeni sledeći pokazatelji: brzina kretanja, vučni otpor, klizanje gusenica, vučna snaga, radni zahvat, specifični otpor zemljišta i učinak. Pored ispitivanja eksploatacionih parametara obavljeno je snimanje strukture radnog vremena. Pored toga date su tehničke karakteristike ispitivanog agregata u rigolovanju. Osnovni zaključak je da rigolovanje angažuje veliku energiju koja se kreće i do 250 kWh/ha za dubine obrade od 72 cm tako da je neophodno posvetiti značajniju pažnju racionalnijim načinima obrade zemljišta pre podizanja višegodišnjih zasada

Aspekti obrade zemljišta u višegodišnjim zasadima

Highly intensive fruit production in both basic and supplemental soil cultivation, along with other agricultural practices was found to impact both growth and fertility of fruit trees. Favourable effects may be expected provided appropriate and timely soil cultivation is employed. Soil cultivation practices range from intensive to completely lacking, which results in the implementation of technical solutions such as machines and tools. The choice depends on the training system, interrow spacing, climate, soil, ...... The aim of the study was to analyze exploitation parameters of the most frequently used rotary hoe („Nardi”, Model ZH/B 145 C). Based on the results obtained, both current and potential machine users may be informed of the advantages and disadvantages of the cultivation. The machine may be used provided working widths amount to 1.45 m and in palmette and slender spindle training systems. Optimal aggregates from the aspect of basic exploitation possibilities need to be analyzed focusing on the financial effect of fruit production.Obrada zemljišta, osnovna i dopunska, u visokointenzivnim voćnjacima, sa ostalim agrotehničkim merama, neposredno utiče na rast i rodnost gajenih voćaka. Navedeni efekti postižu se samo u uslovima pravilne i pravovremene obrade zemljišta. Iskustava u obradi zemljišta kreću se od vrlo intenzivne do potpunog izostavljanja, što ima za posledicu primenu velikog broja tehničkih rešenja mašina i oruđa. Metod izbora polazi od uzgojnog oblika, međurednog rastojanja biljaka, klime, zemljišta... Predmet rada je ispitivanje ekspoatacionih parametara rotofreze za istovremenu rednu i međurednu obradu marke »Nardi« model ZH/B 145 C. Ispitivani tip mašine je veoma zastupljen u našoj voćarskoj praksi. Na osnovu analize dobijenih rezultata, sadašnjim i potencijalnim korisnicima mašine mogu se predočiti prednosti i nedostaci ove varijante obrade zasada. Uzgojni oblik koji dozvoljava upotrebu ove mašine, čiji je radni zahvat od 1,45 m, su svi tipovi Palmeta, Vitko vreteno… Optimizacija složenih sistema značajno ističe i nužnost optimalnog agregata sa aspekta elementarnih eksploatacionih mogućnosti, koje treba posmatrati u interakciji sa finansijskim efektom koji opterećuje proizvodnju u voćnjacima

Sastav lipida fermentisanih kobasica sa dodatkom lanenog ulja

The study examined the fatty acid profile of fermented sausages in which one part of backfat (making up 5%, 7% and 9% of the control treatment formulation) was replaced with flaxseed oil pre-treated with alginate and soy protein isolate. Increase in the content of pre-treated flaxseed oil had a significant effect on the fatty acid profile, which progressively changed with the increase of the oil content: saturated fatty acid content, content of oleic and total monounsaturated fatty acids were significantly lower while the content of α-linolenic acid increased multiple times (15–40 times) with the addition of flaxseed oil. Fermented sausages with flaxseed oil had significantly favorable PUFA/SFA and n-6/n-3 ratios.U ovom ogledu ispitivan je sastav masnih kiselina fermentisanih kobasica kod kojih je deo čvrstog masnog tkiva (ČMT, koje čini 5, 7 i 9% nadeva) zamenjen lanenim uljem pripremljenim kao alginatni gel i emulzijom sa izolatom proteina soje. Zamena dela ČMT-a lanenim uljem imala je značajan uticaj na sastav masnih kiselina koji se progresivno menjao sa povećanjem sadržaja ulja: sadržaj zasićenih masnih kiselina, oleinske i ukupnih mononezasićenih masnih kiselina je značajno smanjen dok je sadržaj α-linolenske kiseline višestruko veći (15–40 puta) kod varijanti s lanenim uljem. Fermentisane kobasice sa lanenim uljem imale su značajno povoljnije odnose PUFA/SFA i n-6/n-3

Senzorni kvalitet fermentisanih kobasica sa dodatkom lanenog ulja tokom skladištenja

The study examined the impact of the replacement of backfat with linseed oil on the sensory characteristics of fermented sausages. The oil was added as alginate gel and as emulsion with soy protein isolate. The content of the oil replacing backfat was around 5%, 7% and 9% of the batter. Increase the oil content and the manner of preparation did not influence the appearance and surface color. As for cut appearance, color, odor and taste grades declined progressively with the increase in the oil content. During storage, changes were mostly of the same intensity as in control. Content of linseed oil higher than 5% can negatively impact the acceptability of fermented sausages.U ovom ogledu ispitivan je uticaj zamene čvrstog masnog tkiva (ČMT) lanenim uljem na senzorna svojstva fermentisanih kobasica. Ulje je dodato kao alginatni gel i kao emulzija sa izolatom proteina soje. ČTM je zamenjen tako da u nadevu sadržaj ulja bude oko 5%, 7% i 9%. Povećanje zamenjenog dela ČMT-a i način pripreme generano nisu uticali na izgled na preseku i boju površine. Međutim, u pogledu izgleda na preseku, boje na preseku, mirisa i ukusa ocene su sa povećanjem sadržaja ulja progresivno bile manje. Tokom skladištenja promene uglavnom nisu bile većeg intnziteta od kontrole. Veći sadržaj lanenog ulja od 5% u nadevu feremnisanih kobasica može negativno da utiče njihovu prihvatljivost