The first step of viral infection requires the binding of the viral attachment protein to cell surface receptors. Following binding, viruses penetrate the cellular membrane to deliver their genome into the host cell. For enveloped viruses, which have a lipid bilayer that surrounds their nucleocapsids, entry into the host cell requires the fusion of viral and cellular membranes. This process is mediated by viral glycoproteins located on the surface of the virus. For many enveloped viruses, such as influenza, Ebola, and human immunodeficiency virus, the fusion protein is responsible for mediating both attachment to cellular receptors and membrane fusion. However, paramyxoviruses are unique among fusion promoting viruses because their receptor binding and fusion activities reside on two separate proteins. This unique distribution of functions necessitates a mechanism by which the two proteins can transmit the juxtaposition of the viral and host cell membranes, mediated by the attachment protein (HN/H), into membrane fusion, mediated by the fusion (F) protein. This mechanism allows for paramyxoviruses to gain entry into and spread between cells, and therefore, is an important aspect of virus infection and disease progression. Despite the conservation of receptor binding activity among members of the Paramyxovirinaesubfamily, for most of these viruses, including Newcastle disease virus (NDV), heterologous HN proteins cannot complement F in the promotion of fusion; both the HN and F proteins must originate from the same virus. This is consistent with the existence of a virus-specific interaction between the two glycoproteins. Thus, one or more domains on the HN and F proteins is thought to mediate a specific interaction between them that is an integral part of the fusion process. Therefore, the primary focus of this thesis is the identification of the site(s) on HN that directly contacts F in the HN-F interaction. The ectodomain of the HN protein consists of a stalk and a terminal globular head. Analysis of the fusion activity of chimeric paramyxovirus HN proteins indicates that the stalk region of HN determines its F protein specificity. The first goal of this research was to address the question of whether the stalk not only determines F-specificity, but does so by directly mediating the interaction with F. To establish a correlation between the amount of fusion and the extent of the HN-F interaction, a specific and quantitative co-immunoprecipitation assay was used that detects the HN-F complex at the cell surface. As an initial probe of the role of the HN stalk in mediating the interaction with F, N-glycans were individually added at several positions in the region. N-glycan addition at positions 69 and 77 in the stalk specifically and completely block both fusion and the HN-F interaction without affecting either HN structure or its other activities. However, though they also prevent fusion, N-glycans added at other positions in the stalk also modulate activities that reside in the globular head of HN. This correlates with an alteration of the tetrameric structure of the protein as indicated by sucrose gradient sedimentation analyses. These additional N-glycans likely indirectly affect fusion, perhaps by interfering with changes in the conformation of HN that link receptor binding to the fusion activation of F. To address the issue of whether N-glycan addition at any position in HN would abolish fusion, an N-glycan was added in another region at the base of the globular head of HN (residues 124-152), which was previously predicted by a peptide-based analysis to mediate the interaction with F. HN carrying this additional N-glycan exhibits significant fusion promoting activity, arguing against this site being part of the F-interactive domain in HN. These data support the idea that the F-interactive site on HN is defined by the stalk region of the protein. Site-directed mutagenesis was used to begin to explore the role of individual residues in the stalk in the interaction with F. The characteristics of the F-interactive domain in the stalk of HN are that it is a conserved motif with enough sequence heterogeneity to account for the specificity of the interaction. One such region that meets these requirements is the intervening region (IR) (residues 89-95); a non-helical domain situated between two conserved heptad repeats. Several amino acid substitutions for a completely conserved proline residue in this region impair not only fusion and the HN-F interaction, but also decrease neuraminidase activity in the globular domain and alter the structure of the protein, suggesting that the substitutions indirectly affect the HN-F interaction. Substitutions for L94 also interfere with fusion, but have no significant effect on any other HN function or its structure. Amino acid substitutions at two other positions in the IR (A89 and L90) also modulate only fusion. In all cases, diminished fusion correlates with a decreased ability of the mutated HN protein to interact with F at the cell surface. These findings indicate that the IR is critical to the role of HN in the promotion of fusion and are consistent with its direct involvement in the interaction with the homologous F protein. These are the first point mutations in the HN protein for which a correlation has been demonstrated between the extent of the HN-F interaction and the amount of fusion. This argues strongly that the co-IP assay is an accurate reflection of the HN-F interaction. The second goal of this research was to address the HN-F interaction from the perspective of the F protein by investigating the relationship between receptor binding, the HN-F interaction, and fusion using a highly fusogenic form of the F protein. It has previously been shown that an L289A substitution in NDV F eliminates the requirement for HN in the promotion of fusion and enhances HN-dependent fusion above wild-type (wt) levels. Here, it was shown that the HN-independent fusion exhibited by L289A-F in Cos-7 cells cannot be duplicated in BHK cells. However, when L289A-F is co-expressed with wt HN, enhanced fusion above wt levels is observed in BHK cells. Additionally, when L289A-F is co-expressed with IR-mutated HN proteins previously shown to promote low levels of fusion with wt F, a 2.5-fold increase in fusion was observed. However, similar to wt F, an interaction between L289A-F and the IR-mutated HN proteins was not detected. These results imply that the attachment function of HN, as well as the conformational change in L289A-F, are necessary for the enhanced level of fusion exhibited by HN proteins co-expressed with L289A-F. Indeed, two MAbs detected a conformational difference between L289A-F and the wt F protein. These findings support the idea that the L289A substitution converts F to a form that is less dependent on an interaction with HN for conversion to the fusion-active form. The last goal of this research was to address the cellular site of the HN-F interaction, still a controversial issue based on conflicting data from studies of different paramyxoviruses, using various approaches. This is a particular point of interest, as it speaks to the mechanism by which the HN-F interaction regulates fusion. Thus, NDV HN and F were successfully retained intracellularly with a multiple arginine or KK motif, respectively. The results of Endoglycosidase H resistance and F cleavage studies indicate that the mutated proteins, HN-ER and F-ER, are retained in a compartment prior to the medial-Golgi apparatus and that they are unable to interact with a high enough affinity to co-retain or even cause reduced transport of their wt partner glycoproteins. This is consistent with the HN-F interaction occurring at the cell surface, possibly triggered by receptor binding. In conclusion, this thesis presents evidence to argue that the IR in the stalk of the NDV HN protein directly mediates the interaction with the F protein that is necessary for fusion. Overall, the data presented in this thesis extend the current knowledge of the mechanism by which the paramyxovirus attachment protein can trigger the F protein to initiate membrane fusion. A clear understanding of this process has the potential to identify new anti-viral strategies, such as small molecule inhibitors, aimed at controlling paramyxovirus infection by interfering with early steps in the virus infection cycle.

Heavy chain isotype switch recombination is preceded by the appearance of RNA initiating 5' of the specific switch region which will undergo recombination. In an effort to understand the potential function of germline transcripts in switch recombination and the degree to which the regulation of germline transcripts correlates with the regulation of switching, we studied this process in the murine B-lymphoma cell line I.29μ, which in the presence of bacterial lipopolysaccharide (LPS) switches primarily to IgA and less frequently to IgE. Levels of α-germline transcripts initiating upstream of α switch (Sα) sequences are elevated in clones of this line which switch well as compared to clones which switch less frequently. TGFβ1 has been shown to increase α-germline transcripts and switching to IgA expression in LPS-stimulated murine splenic B-cells. We now demonstrate in I.29μ cells that TGFβ also increases switching to IgA and increases the level of α-germline transcripts 5 to 9 fold. Nuclear run-on analysis shows that this increase is at the level of transcription. Thus, TGFβ appears to direct switching to IgA by inducing transcription from the unrearranged Sα- CαDNA segment. Germline α RNA is quite stable in I.29μ cells, having a half life of about 3 to 5 hours, and we find only slight stabilization in the presence of TGFβ. Levels of ε-germline transcripts are not increased by TGFβ . IL-4, which modestly increases switching to IgA in I.29μ cells, slightly increases trancription of α-germline RNA. However, we present evidence suggesting that endogenously produced IL-4 may also act at additional levels to increase switching to IgA. IFNγ, which reduces IgA expression in these cells, also reduces the level of α-germline transcripts. IFNγ also reduces the level of ε-germline transcripts induced by IL-4. Our results support the hypothesis that the regulation of transcription of particular switch sequences by cytokines in turn regulates the specificity of recombination. In studies aimed at identifying other signalling pathways that promote class switching, we discovered that inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) increase lipopolysaccharide (LPS)-induced switching to IgA in the B cell lymphoma I.29μ and to IgG1 in LPS + IL-4-treated splenic B cells. PARP, which binds to and is activated by DNA strand breaks, catalyzes the removal of ADP-ribose from NAD+ and poly(ADP-ribosylation) of chromatin-associated acceptor proteins. This enzyme is believed to function in cellular processes involving DNA strand breaks as well as in modulating chromatin structure. In I.29μ cells, PARP inhibitors increase IgA switching by day 2 and cause a 5-fold average increase in switching on day 3 as assayed by immunofluorescence microscopy. The PARP inhibitor, nicotinamide, also causes a reduced intensity of hybridization of Cμ and Cα specific probes to genomic DNA fragments containing the expressed VDJ-Cμ and the unrearranged Sα - Cα segments, respectively, indicating that PARP inhibition increases rearrangment of these fragments. Induction of switching by PARP inhibitors is not mimicked by treatment with cAMP analogs or reduced by inhibitors of protein kinase A (PKA). Induction of switching by PARP inhibitors does not appear to involve increased levels of transcription of the unrearranged Cα gene, although TGFβ is required for optimal induction by PARP inhibitors, consistent with a requirement for transcription of the unrearranged CH gene. PARP inhibitors do not overcome the requirement for endogenously produced IL-4.

This diploma thesis deals with dynamic of accumulation of non-structural carbohydrates and activity of Rubisco enzyme at elevated concentration of CO2 on beech (Fagus sylvatica L.). Three years old seedlings of beech were cultivated in minisphere with ambient (385 µmol•mol-1, variant A), and with elevated concentration CO2 (700 µmol•mol-1, variant E) for four months. In every variant the first half of plants was fertilized by nitrogen (variant N+) and the second half was control (variant N-). Plants used for experiment were at first adapted for darkness for 12 hours. Subsequently tested leaves were cut off, leafstalk including short segment of branch (approximately 1 cm) was inserted into 0.7 M solution of sucrose (variant S) or water (variant V) and exposed to radiation 200 mol•m-2•s-1 for 0, 30, 60, 120, 180 minutes. Then leaf area and fresh mass of leaf blade were established, samples were fixed in liquid nitrogen and stored in deep freezer to analysis in –70 °C. Rubisco content was determined by SDS-PAGE method, Rubisco activity spectrofotometrically and content of non-structural carbohydrates by anthrone method and HPLC method. Rubisco content was significantly lower in the N- variant than in N+ variant. Rubisco content was also significantly lower in E than in A variant, which is an evidence of down-regulation. Rubisco activity is moderately stimulated at E variant with time, but differences between variant A and E are not statistically significant. Influence of sucrose feeding to Rubisco activity was not proved. Significant differences were detected by anthrone method in non-structural carbohydrates content between variants S and V, but not between variants A and E. Statistically significant increase of sucrose content with time was detected by HPLC method at variant AS, but not at variant ES.

No description available.

Resumo: O objetivo deste trabalho foi avaliar respostas do capim-tifton 85 a doses de nitrogênio (N) associadas a doses e fontes de boro (B). Em casa de vegetação, conduziu-se um experimento fatorial 3 ' 3 ' 3 {três doses de N (0, 100 e 200 mg kg-1), três doses de B (0, 1,3 e 2,6 mg kg-1) e três fontes de B [colemanita fundida ao termofosfato (CF), colemanita em mistura com termofosfato (CM) e ácido bórico (H3BO3)]} em delineamento inteiramente ao acaso. As plantas cresceram em vasos preenchidos com amostra de um Latossolo Vermelho distrófico. Elas foram avaliadas em três ciclos de crescimento. O N foi aplicado no início de cada ciclo e o B apenas antes do plantio. A aplicação de N aumentou a massa seca da parte aérea e o número de perfilhos do capim nos três ciclos. A aplicação de B, isolada ou associada à de N, não teve influência nesses atributos, embora o solo tivesse baixo teor de B disponível. O suprimento de N aumentou também os teores de carboidratos de reserva na raiz da planta nos dois ciclos em que foram avaliados (primeiro e terceiro). O suprimento de B aumentou o teor desses carboidratos na base do caule no primeiro ciclo. Os fornecimentos de N e B aumentaram seus respectivos teores na parte aérea do capim no três ciclos, exceto no caso do teor de B no terceiro ciclo. Todas as fontes de B aumentaram o teor de B disponível no solo, mas a CM e o H3BO3 aumentaram mais do que a CF. O efeito mais evidente da aplicação conjunta de N e B ocorreu no acúmulo de B na parte aérea da planta, aumentando mais com a combinação das duas aplicações. O teor inicial de B no solo (0,15 mg dm-3) foi suficiente para atender as necessidades do capim-tifton 85 nos três ciclos de crescimento. / Abstract: The objective of this work was to evaluate responses of Tifton 85 bermudagrass to nitrogen (N) rates associated with boron (B) rates and sources. In greenhouse, it was carried out a factorial experiment 3 ' 3 ' 3 {three N rates (0, 100 and 200 mg kg-1), three B rates (0, 1.3 and 2.6 mg kg-1) and three B sources [colemanite fused to termophosphate (CF), colemanite in mixture with termophosphate (CM) and boric acid (H3BO3)]} in complete randomized design. The plants grew in pots filled with sample of a Typic Haplustox. They were evaluated during three growth cycles. The N was applied at the beginning of each cycle and the B just before the planting. The N application increased both top dry mass and tiller number of the grass in the three cycles. The B application, isolated or associated with N application, did not have influence on these attributes, though the soil had low available B content. The N supply increased reserve carbohydrates contents in plant root in the two cycles in which they were evaluated (first and third). The B supply increased the content of these carbohydrates in the stem base in the first cycle. The N and B furnishings increased their respective contents in top grass in the three cycles, except in the case of the B content in the third cycle. All the sources increased the available B content in soil, but CM and H3BO3 increased more than CF. The most evident effect of the N and B applications together occurred in the B accumulation in top plant, increasing more with the combination of the two applications. The initial B content in soil (0.15 mg dm-3) was enough to attend necessities of Tifton 85 bermudagrass in the three growth cycles. / Orientador: Edson Luiz Mendes Coutinho / Coorientador: Edemo João Fernandes / Banca: Mara Cristina Pessôa da Cruz / Banca: Cassio Hamilton Abreu Junior / Banca: Takashi Muraoka / Banca: Renato de Mello Prado / Doutor

Potato is one of the most important agricultural crops and there is an attempt to increase and improve yields of tubers, among other things, by elucidation of the mechanisms that regulate the process of tuber induction. Potato tuberization is a morphogenetic process in which the tubers are formed from the underground parts of the stem - stolons. The correct timing of this process is controlled by a complex regulatory network and influenced by many internal and external factors. Under favourable conditions, an inductive signal is generated in the leaves and it is transported to the stolon by a "phloem information superhighway" driven by carbohydrates flow. The signal triggers cell division, expansion, and changes in the cell growth orientation in the stolon. The development of tubers is influenced by number of biochemical and morphological processes driven by a regulatory network of genes that are expressed in different parts of plants. This work was focused on Solanum tuberosum, Lada cultivar and its derived D69 mutant line with lacking isoform of manganese-stabilizing protein (MSP), which is so far the only dissimilarity identified under all tested conditions. I aimed to map the processes related to the production of carbohydrates in leaves (photosynthetic characteristics - rate of photosynthesis...

Heavy metal contamination significantly reduces crop yields, causing serious problems in agriculture and having a major impact on human health if these contaminants enter the food chain. Understanding the mechanisms of plant responses could help to increase their resistance to heavy metals as well as their potential use in phytoremediation. Carbohydrates play an important role in plant growth and development as well as in defense reactions. This work summarizes the results of four publications focused on the effects of arsenic and thorium on antioxidant mechanisms in tobacco plants and horseradish root crops. Attention is paid, among other things, to the dynamics of sugar contents, which are potentially important molecules involved in the fight against oxidative stress. The first publication summarizes arsenic effects on plant physiological parameters, focusing on arsenic tolerance-enhancing mechanisms as well as summarizing the ability of plants to cope with arsenic-induced oxidative and nitrosative stress. Emphasis was placed on, among other things, a topic that was unjustly neglected in previous publications - i.e. carbohydrate metabolism. Further work was already experimental and dealt with the study of arsenic as a trigger of oxidative stress in the root culture of horseradish and...

Variations in composition and disappearance of nutrients in dairy cattle feeds are dictated by ingredients, methods of processing, storage while milk production levels depend on the animal, environmental factors and largely on pools of available carbohydrates, proteins, vitamins and minerals in the concentrate feeds. There is a wide variety of concentrates for dairy cattle on the formal and informal markets and dairy farmers need to be astute in selecting feeds appropriate for specific production periods and animals to sustain their businesses. Composition of nutrients displayed on concentrate containers is however inadequate for in-depth assessment of products. This study determined nutrient composition, rumen dry matter disappearance and microbial colonization on residual substrate on commercial concentrate feeds and simulated total mixed rations for dairy calves, heifers, lactating and dry cows based on common feeding guidelines. Equivalent feeds for each herd group were obtained from three suppliers in the formal markets in Gauteng province of South Africa, making a total of twelve. An analysis of the data on container labels for the herd groups displayed similar feed values, as also reflected on the recommendation Tables of Act 36: Feeds and Fertilizer bill 1947 of South Africa. / Agriculture, Animal Health and Human Ecology / M.Sc. (Agriculture)

Carrier mediated nutrient import is vital for cell and tissue homeostasis. Structural insights of carrier mediated transport, particularly the human glucose transporter GLUT1, are essential for understanding the mechanisms of human metabolic disease, and provide model systems for cellular processes as a whole. GLUT1 function and expression is characterized by a complexity unexplained by the current hypotheses for carrier-mediated sugar transport (9). It is possible that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae(RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1) to characterize its functional properties. Identical protein sequences generated different kinetic parameters when expressed in RE700A yeast, erythrocytes, and HEK293 cells. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific. Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 sugar export site. Paradoxically, very low concentrations of these inhibitors produce a modest stimulation of sugar transport (16). This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, intracellular binding sites for e1 ligands CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogs, asks what structural features of exit site ligands determine binding site affinity and cooperativity. Our findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of GLUT1 quaternary structure and evaluate the major determinants of ligand binding affinity and cooperativity. Cytochalasin B (CB) inhibits GLUT1 substrate transport at or near the endofacial sugar binding site. N-bromosuccinamide analysis combined with 3H-CB photolabeling implicates the region between Trp388 and Trp412 in ligand binding. Although its structure has been modeled(5), the specific residues comprising the sugar binding site are unknown. A series of alanine point mutants were made, and mutant protein 2-deoxy glucose transport was tested in the presence of increasing [CB]. Arg126Ala and Cys421Ala GLUT1 mutations altered CB affinity but were determined not to be in the e1 site. The Arg400Ala mutation decreased binding affinity for CB, and may comprise part of the e1 binding site. Because point mutations were individually insufficient to abrogate CB binding, Trp388 to Trp412 chimeras were made. GLUT1/GLUT4388-412/GLUT1 and GLUT1/GLUT5388-412/GLUT1 chimeras showed moderately less sensitivity to CB inhibition of transport; these amino acids likely comprise regions determinant of CB binding affinity. Furthermore GLUT1/GLUT5388-412/GLUT1 shows enhancement of 2-DG uptake at 50nM CB, but an overall dose response indistinguishable from WT GLUT1. A multisite fit of the data suggested GLUT1/GLUT5388-412/GLUT1 chimera possesses strong first site affinity for CB but slight negative second-site cooperativity. We conclude that point mutants were insufficient to abrogate CB binding and that the Trp388 to Trp412 sequence is necessary for CB binding affinity but is not the sole determinant of inhibition of 2 deoxyglucose uptake by CB. We discuss these results with their implications for structure-function sequence localization of the CB binding site, and by extension, the e1 sugar binding site.

Studies have demonstrated that under conditions of chronic metabolic stress, GLUT1-mediated sugar transport is upregulated at the blood-brain barrier by a number of mechanisms. Although acute metabolic stress has also been shown to increase GLUT1-mediated transport, the mechanisms underlying this regulation remain unclear. This work attempts to explain how GLUT1-mediated sugar uptake is increased during acute metabolic stress, as well as explore the factors involved in this modulation of sugar transport in blood-brain barrier endothelial cells. Glucose depletion, KCN and FCCP were applied to brain microvascular endothelial cell line bEnd.3 in order to induce acute metabolic stress by ATP depletion. Kinetic sugar uptake measurements in combination with qPCR, whole cell lysate western blots, and cell-surface biotinylation were employed to probe for changes in GLUT1-mediated sugar uptake, GLUT1 expression levels, and GLUT1 localization during metabolic stress. Finally, the role of AMP-activated kinase (AMPK) in the bEnd.3 cell response to acute stress was examined using the specific AMPK activator AICAR and inhibitor Compound C. The data presented in this thesis supports the following two conclusions: 1. GLUT1-mediated sugar transport in bEnd.3 cells during acute metabolic stress is increased 3-7 fold due to translocation of intracellular GLUT1 to the plasma membrane, with no change in expression of total GLUT1 protein, and 2. AMPK plays a direct role in modulating increases in GLUT1-mediated sugar transport in bEnd.3 cells during acute metabolic stress by regulating trafficking of GLUT1 to the plasma membrane.