Understanding the origin of protein folds, and the mechanism by which evolution has generated them, is a critically important step on a path towards rational protein design. Modifying existing proteins and designing our own novel folds and functions is a lofty but achievable goal, for which there are many foreseeable rewards. It is believed that modern proteins may have arisen from a primordial set of peptide precursors, which were initially only pseudo-stable or stable only as complexes with RNA, and later were able to self-assemble into multimeric complexes that resembled modern folds. In order to experimentally examine the feasibility of this theory, an attempt was made at reconstructing the evolutionary path of a beta-trefoil. The beta-trefoil is a naturally abundant fold or superfold, possessing pseudo-threefold symmetry, and usually having a sugar-binding function. It has been proposed that such a fold could arise from the triplication of just one small peptide on the order of 40-50 amino acids in length. The evolutionary path of a ricin, a family within the beta-trefoils known to possess a carbohydrate binding function was the chosen template for evolutionary modelling. It was desirable to have a known function associated with this design, such that it would be possible to determine if not only the fold, but also the function, could be reconstructed. A small peptide of 47 amino acids was designed and expressed. This peptide not only trimerized as expected, but possessed the carbohydrate binding function it was predicted to have. In an evolutionary model of the early protein world, the gene for this peptide would undergo duplication and later, triplication, eventually resulting in a completely symmetrical beta-trefoil, which would represent the first modern beta-trefoil fold. Such a completely symmetrical protein was also designed and expressed by triplicating the gene for the aforementioned small peptide. This hypothetical first modern beta-trefoil is: well folded, stable, soluble, and appears to adopt a beta-trefoil fold. Together these results demonstrate that an evolutionary model of early life: that proteins first existed as self-assembling modular peptides, and subsequent to gene duplications or fusions, as what we now recognize as modern folds, is experimentally consistent and not only generates stable structures, but those with function, which of course is a prime requisite of evolution. Moreover the results show that it may be possible to use this modular nature of protein folding to design our own proteins and predict the structure of others.

Plants have many useful substances – and one of them is flavonoids with other phenolic compounds. This is a very big group of biological active compounds. Flavonoids have been referred to as "nature's biological response modifiers" because of their ability to modify the body's reaction to other compounds such as allergens, viruses, and carcinogens. They show anti-allergic, anti-inflammatory, and anti-cancer activity. In addition, flavonoids act as powerful antioxidants, providing remarkable protection against oxidative and free radical damage. As a result, consumers and food manufacturers have become increasingly interested in flavonoids for their healthful properties, especially their potential beneficial role in the prevention of cancer and cardiovascular diseases. Phenolic acids are interesting of their protective role against oxidative damage diseases (coronary heart disease, stroke, and cancers). One of possible sources of flavonoids and phenolic acids – Canadian thick-trefoil (showy trefoil) – Desmodium canadense (L) DC. This plant is not researched as good as many other plants, but there are some works on it. In Lithuania, KUM, there were some research works on Desmodium canadense (L.) DC. There was performed analysis of grass, collected in different phase of vegetation. Also was researched influence of mineral fertilising to amounts of flavonoids and phenolic acids.

Gana dažnai nusilpusiai imuninei sistemai stabilizuoti vartojami augaliniai imunostimuliuojantys preparatai, nes jų poveikyje pasireiškia mažiau pašalinių reakcijų, jie saugesni vartojant didesnėmis dozėmis, todėl yra pranašesni už sintetinius cheminius preparatus. Herpes virusų sukeltoms ligoms gydyti vartojami ne tik cheminiai preparatai, bet ir augaliniai vaistai savo sudėtyje turintys flavonoidų ir fenolkarboksirūgščių. Šiuos junginius kaupia kanadinė jakšūnė (Desmodium canadense (L.) DC.). Darbo tikslas – ištirti ketvirtųjų – penktųjų auginimo metų kanadinės jakšūnės (Desmodium canadense (L.) DC.) biologinius savumus Lietuvos klimatinėmis sąlygomis ir nustatyti besikaupian��ių veikliųjų junginių dinamiką vegetacijos eigoje. Buvo nustatyti kanadinės jakšūnės antžeminės dalies augimo ypatumai ketvirtaisiais ir penktaisiais auginimo metais. Ištirta Desmodium canadense (L.) DC. požeminės dalies vystymosi dinamika vaizdinės analizės metodu. Nustatytas kanadinės jakšūnės sėklų daigumas pritaikant skarifikavimo ir stratifikavimo metodus. Atlikta Desmodium canadense (L.) DC. fitocheminė analizė efektyviosios skysčių chromatografijos metodu (ESC) ir nustatyti besikaupiančių veikliųjų junginių kiekiai bei jų dinamika vegetacijos eigoje. Gausiausia veikliųjų junginių susikaupia žydėjimo pradžioje. Kanadinės jakšūnės žolėje tuo laikotarpiu penktaisiais vegetacijos metais rasta 1,38  flavonoidų ir fenolkarboksirūgščių, o lapuose – 2,1  šių junginių. Pagrindiniai veiklieji... [toliau žr. visą tekstą] / Nowadays a lot of people use herbal drugs as immunity stimulating preparations, because they cause less side-effects and are safer in high doses. That’s why they are superior to chemical drugs. Plants have many useful substances – and one of them are flavonoids with other phenolic compounds. One of possible sources of flavonoids and phenolic acids – Showy tick trefoil – Desmodium canadense (L.) DC. The aim of work is to determine biological features of Showy tick trefoil of fourth and fifth vegetation year in Lithuanian climate conditions and to estimate dynamics of the active compounds in different phases of vegetation. There were determined biological features of overground part of plant of fourth and fifth vegetation year. The underground part of Desmodium canadense (L.) was examined by visual analysis method. Seeds of Showy tick trefoil were investigated using scarification and stratification methods. Phytochemical analysis was done by using High pressure liquid chromatography (HPLC). There were determined flavonoids, phenolic acids and their amounts in plant material, collected in different phases of vegetation. The most active compounds plant accumulates in the beginning of flowering. In this period in herb of Desmodium canadense (L.) was found 1,38 flavonoids and phenolic acids, while in leaves – 2,1. The most important active compounds are apigenin, luteolin and their glycosides – orientin, vitexin, isovitexin, also rutin.

Kanadinė jakšūnė nėra gerai ištyrinėtas vaistinis augalas, todėl buvo tikslinga atlikti Lietuvoje išaugintos žaliavos fitocheminius tyrimus augalo veikliųjų junginių sudėčiai nustatyti. Sukaupti duomenys gali būti naudojami iš kanadinės jakšūnės gaminamų preparatų standartizacijai ir naujų, dar nenustatytų farmakologinių poveikių paieškai. Tyrimo objektas ir metodai: kanadinės jakšūnės žaliava ištirta efektyviosios skysčių chromatografijos metodu. Darbo tikslas ir uždaviniai: atlikti kanadinės jakšūnės kaupiamų flavonoidų ir fenilpropanoidų kokybinės ir kiekybinės sudėties tyrimą bei nustatyti, kokiame augalo organe ir kokiu vegetacijos tarpsniu jų kaupiama daugiausiai. Taip pat nustatyti dominuojančius veikliuosius junginius žolėje ir atskiruose organuose bei atlikti palyginamąja ataugusios žaliavos analizę. Ištirti kanadinės jakšūnės žolėje ir lapuose sukauptų flavonoidų suminių kiekių dinamiką skirtingais vegetacijos tarpsniais antraisiais – septintaisiais augimo metais ir pateikti žaliavos rinkimo rekomendacijas bei nustatyti, ar augalo amžius turi įtakos kaupiamų veikliųjų junginių kiekiui. Išvados: Daugiausiai veikliųjų junginių visais vegetacijos tarpsniais susikaupia kanadinės jakšūnės lapuose, išskyrus žydėjimo tarpsnį, kai didžiausias nustatytų veikliųjų junginių kiekis randamas žieduose. Lapuose ir žolėje daugiausiai veikliųjų junginių šeštaisiais augimo metais sukaupta butonizacijos tarpsniu, o stiebuose – vegetacijos pabaigoje. Septintaisiais augimo metais... [toliau žr. visą tekstą] / Showy tick-trefoil is not widely explored medical plant. Therefore it is reasonable to carry out a phytochemical research for the plant which was grown in Lithuania in order to identify its composition of active compounds. Accumulated information of analysis can be useful in standardization of pharmaceutical preparations from showy tick-trefoil, also in researching previously unidentified pharmacological effects. Object and methods: raw material of showy tick-trefoil was examining by methods of high pressure liquid chromatography. Objectives: To perform accumulated flavonoids and fenilpropanoids quality and quantity analysis on showy tick-trefoil and identify in which organ and which vegetation period they are mostly compiled; to identify dominating active compounds in herb and individual organs; to make a comparative analysis of outgrowth raw material. Also to investigate total amounts of compiled flavonoids in showy tick-trefoil herb and leaves in different vegetation periods from second till seventh year of herb growing; to give recommendations for picking raw material. Finally to indicate whether the age of the plant has any influence for quantity of active compounds. Findings: The biggest amount of active compounds in every vegetation period is concentrated in leaves, except blooming period. In the sixth year of growing most of active compounds in leaves and in herb are founded during budding period, in stems – during the end of vegetation. During seventh year of growing... [to full text]

Fodder from lasting grass growths serves from 80% to fed ruminants. Therefore is setting high emphasis on quality fodder from trifolium, grass and trifolium-grass growths. On this account fit itrodukcion try in these growths imply at full blast useful kinds plants (trifolium), which have improving effects on fodder. Once of these improving products is also Lathyrus pratensis. His introduction increases substitution legumin in lasting meadow growths and share rhizobial nitrogen on production knows - nose fodder and nutritive valuable degestible nitrogen factory materials. Occurrence Lathyrus pratensis is impressed with type herbal growth and his differential management.

La décision du traitement adjuvant du cancer du sein est souvent difficile. Actuellement, il est nécessaire de développer de nouveaux outils pronostiques et prédictifs de sensibilité aux traitements. TFF1 (Trefoil Factor 1) et TFF3 (Trefoil Factor 3) sont deux petites protéines sécrétées dont l'expression est induite par l'estradiol dans les cellules épithéliales de cancer du sein. Étudiés séparément, leur intérêt pronostique est controversé et des résultats contradictoires ont été reportés. L'objectif de notre étude était d'évaluer l'intérêt pronostique du dosage combiné de TFF1 et de TFF3 dans une série prospective de tumeurs du sein. En accord avec la littérature, nous avons trouvé que l'expression de TFF1 et/ou de TFF3 était spécifique des tumeurs exprimant le récepteur aux estrogènes ("luminal-like"). Contrairement aux résultats d'études précédentes,TFF1 et TFF3 étaient exprimés indépendamment l'un de l'autre dans certaines tumeurs. De manière intéressante, la présence conjointe de TFF1 et de TFF3 dans les tumeurs a été associée à des éléments de pronostic péjoratif, discriminant un sous-groupe de plus haut risque au sein des cancers du sein luminaux. Par conséquent, les tumeurs TFF1 +/TFF3+ pourraient être candidates à une thérapie adjuvante. TFF1 et TFF3 sont sécrétés sous différentes formes moléculaires, comme monomères, homodimères ou hétérodimères. Ces différentes formes moléculaires sont supposées avoir différentes fonctions. En utilisant la PLA (Proximity Ligation Assay), nous avons montré la présence de l'hétérodimère TFF1/TFF3 dans des échantillons de cancer du sein double-positifs.Ces données suggèrent que l'hétérodimère TFF1/TFF3 pourrait participer au phénotype plus agressif des tumeurs doubles positives. / The breast cancer adjuvant treatment decision is often difficult. Currently, it is necessary to develop new prognostic and predictive markers in oncology. Trefoil factor 1 (TFF1) and trefoil factor 3 (TFF3) are two related small secreted proteins induced by estradiol in mammary epithelial cancer cells. Studied in isolation, their value as prognostic markers was controversial and contradictory results were reported. The aim of our study was to evaluate the prognostic value of combined TFF1 and TFF3 dosages in a prospective breast cancer series. ln agreement with the literature, we found that TFF1 and/or TFF3 expressions were specific to estrogen-receptor positive tumours ("luminal-like"). ln contrast with previous reports, TFFi and TFF3 were expressed independently from each other in some tumors. Of interest, the presence of bath TFF1 and TFF3 was associated with poor prognosis elements, distinguishing a high-risk subtype within luminal breast cancers.Therefore, TFF1 +/TFF3+ breast cancers should be considered for adjuvant therapy. TFF1 andTFF3 are secreted under distinct molecular forms, monomers, homodimers and heterodimers.These distinct molecular forms are supposed to have different functions. By using proximity ligation assay (PLA), we found TFF1/TFF3 heterodimers in double-positives cancer samples. These data suggest that TFF1/TFF3 heterodimer may contribute to the aggressive phenotype observed in double positive tumors.

Three studies applicable to organic management and cultivation of birdsfoot trefoil (BFT, Lotus corniculatus L.) are presented here. The first is a histological analysis of lignification in BFT stems that supports recommendations to harvest BFT at approximately 6 weeks of regrowth, or early bloom. Lignification decreases digestibility and is correlated with high shear force required to break BFT stems. The sixth internodes from the base of 10 BFT and two alfalfa plants were sampled during 15 weeks of midsummer regrowth. The lignified xylary ring reached its maximum radial width by 7.5 weeks of regrowth, and full bloom was reached by 6.5 weeks of regrowth. The second study evaluated establishment of BFT, which competes poorly with weeds and produces low yields during establishment. At an organic, irrigated site in northern Utah the effects of autumn vs. spring seeding, seeding rates of 3, 7 20, and 34 kg pure live seed (PLS) ha-1 and use of a companion crop on 3 years of annual yields and foliar cover of mature stands were determined. First year yields increased linearly with seeding rate (P≤0.05). Use of a companion crop reduced first year yields, and did not reduce weed cover. Autumn seeding of BFT alone, following harvest of a summer crop, is recommended for irrigated production. The final study evaluated BFT establishment on five organic dairy farms in souther Idaho and northern Utah. Participating producers broadcast seeded 4-ha BFT pastures in the fall at a rate of 25 kg PLS ha-1. All farms achieved high BFT density, but only two farms had higher BFT than weed density. These two farms also had high BFT cover the spring following autumn seeding, and their pastures produced 6000 to 7600 kg of dry matter ha-1 by 20 June 2012 and supported grazing for the remainder of the summer. Establishment was enhanced by crop rotation and sprinkler irrigation.

My objectives were to determine if nutritional benefits occur when animals are offered foods with compounds -- alkaloids, saponins, and tannins - that are potentially complementary. I hypothesized that food intake and digestibility increase when lambs consume plants such as alfalfa ALF that contain saponins or birdsfoot trefoil (BFT) that contain tannins when the basal diet is endophyte-infected tall fescue (TF) or reed canarygrass (RCG) both of which contain alkaloids. I predicted that the nutritional status of lambs would be enhanced if basal diets of alkaloid-containing grasses were supplemented with ALF or BFT. Lambs fed a basal diet of either endophyte-infected TF or RCG ate more food and consequently digested more dry matter, energy and nitrogen when supplemented with ALF or BFT. Lambs ingested more dry matter and digested more nutrients when fed a basal diet of RCG than one of TF, and supplementing with ALF and BFT was more beneficial for lambs fed TF than for lambs fed RCG. Increased intake of digestible nutrients was due to greater intake when lambs were offered more than one food, not due to an increase in digestibility. In pen trials meant to complement the field trials, lambs were offered an alkaloid-containing (either gramine or 5-methoxy-N,N-dimethyltryptamine) total mixed ration and supplemented with a food that contained saponins or tannins. All rations were isocaloric (3.3Mcal/kg) and isonitrogenous (14% CP). Lambs fed a ration with either alkaloid and offered a food containing saponin digested approximately the same amount of dry matter, energy, nitrogen, and NDF as lambs not offered saponin. When lambs were fed a ration with either alkaloid and supplemented with food that contained tannins, tannin consumption adversely affected dry matter, energy, and NDF digestibility, but lambs offered food with tannins increased dry matter intake, and as a result, they digested the same amount of dry matter, energy, and NDF as lambs not offered the food with tannins. Lambs offered tannin digested and retained more nitrogen than lambs not offered tannin. These findings indicate a nutritional advantage for sheep eating mixtures as opposed to monocultures of foods with different profiles of secondary compounds and nutrients.

Rising fertilizer prices have led a return to the use of grass-legume mixtures to reduce N costs and improve pasture productivity. The objective of this study was to determine optimal species combinations of binary grass-legume mixtures to improve forage production and pasture nutritive value in irrigated pastures of the Intermountain West. The study was conducted at the Utah State University Intermountain Pature Research Facility near Lewiston, UT. Tall Fescue (TF), Orchardgrass (OG), and meadow brome (MB) were grown with alfalfa (ALF), birdsfoot trefoil (BFTF), and cicer milkvetch (CMV) in legume-grass mixes and monocultures at planting ratios of 25:75, 50:50, 75:25. Grass monocultures were fertilized with 0 (0 N), 67 (67 N), or 134 kg N ha-1 (134 N). Forage was harvested four times each season during 2011-2012. Forage of the mixtures and monocultures from the first and third harvests was analyzed for crude protein (CP) and neutral-detergent fiber (NDF). Average forage production of the unfertilized TF, MB, and OG monocultures was 11.03, 9.76, and 8.10 Mg ha-1, respectively. TF-ALF, OG-ALF, and MB-ALF grass-legume mixes averaged 24.0, 35.0, and 41.0% higher forage production than their respective unfertilized grass monocultures. The grass-legume mixtures with the highest CP were MB-ALF 159, TF-ALF 159, and TF-OG-159 g kg-1 and average 59, 43 and 51% higher than their respective unfertilized grass monocultures. Likewise, the mixtures with the lowest NDF were OG-ALF 453 g kg-1, OG-BFTF 469 g kg-1, and MB-ALF 480 g kg-1. These mixtures had 10, 7, and 18% lower NDF than their respective unfertilized grass monocultures. Individual harvests had similarly higher yields and CP, with lower NDF for the mixtures than the unfertilized grass monocultures. The grass-legume mixture with the 50:50 planting ratio were most productive and had high forage quality. The grass-legume mixtures had similar forage production as the grass monocultures at 134 kg N ha-1. The grass-legume mixtures also had higher CP and lower NDF than the grass monocultures. Cicer milkvetch did not perform well in irrigated pastures. Grass-legume mixtures with ALF and BFTF can be used to replace commercial N while increasing forage nutritive value.

Thiostrepton (TS: TS; C72H85N19O18S5) is a thiazoline antibiotic that is effective against Gram-positive bacteria and the malarial parasite, Plasmodium falciparum. Tight binding of TS to the bacterial L11-23S ribosomal RNA (rRNA) complex of the large 50S ribosomal unit inhibits protein biosynthesis. The TS producing organism, Streptomyces azureus, biosynthesizes thiostrepton-resistance methyltransferase (TSR), an enzyme that uses S-adenosyl-L-methionine (AdoMet) as a methyl donor, to modify the TS target site. Methylation of A1067 (Escherichia coli ribosome numbering) by TSR circumvents TS binding. The S. azureus tsr gene was overexpressed in E. coli and the protein purified for biochemical characterization. Although the recombinant protein was produced in a soluble form, its tendency to aggregate made handling a challenge during the initial stages of establishing a purification protocol. Different purification conditions were screened to generate an isolation protocol that yields milligram quantities of protein with little aggregation and sufficient purity for crystallographic studies. Enzymological characterization of TSR was carried out using an assay to monitor AdoMet-dependent ([methyl-3H]-AdoMet) methylation of the rRNA substrate by liquid scintillation counting. During the optimization of assay, it was found that, although this method is frequently employed, it is very time and labour intensive. A scintillation proximity assay was investigated to evaluate whether it could be a method for collecting kinetic data, and was found that further optimization is required. Comparative sequence analysis of TSR has shown it to be a member of the novel Class IV SpoUT family of AdoMet-dependent MTases. Members of this class possess a non-canonical AdoMet binding site containing a deep trefoil knot. Selected SpoUT family proteins were used as templates to develop a TSR homology model for monomeric and dimeric forms. Validation of the homology models was performed with structural validation servers and the model was then used as the basis of ongoing mutagenesis experiments. The X-ray crystal structure of TSR bound with AdoMet (2.45 Å) was elucidated by our collaborators, Drs. Mark Dunstan and Graeme Conn (University of Manchester). This structure confirms TSR MTase’s membership in the SpoUT MTase family with a deep trefoil knot in the catalytic domain. The AdoMet bound in the crystal structure is in an extended conformation not previously observed in SpoUT MTases. RNA docking simulations revealed some features that may be relevant to binding and recognition of TSR to the L11 binding domain of the RNA substrate. Two structure-activity studies were conducted to investigate the TS-rRNA interaction and TS solubility. Computational analyses of TS conformations, molecular orbitals and dynamics provided insight into the possible modes of TS binding to rRNA. Single-site modification of TS was attempted, targeting the dehydroalanine and dehydrobutyrine residues of the antibiotic. These moieties were modified using the polar thiol, 2-mercaptoethanesulfonic acid (2-MESNA). Similar modifications had been previously used to improve solubility and bioavailability of antibiotics. The resulting analogue was structurally characterized (NMR and mass spectrometry) and showed antimicrobial activity against Bacillus subtilis and Staphylococcus aureus.