Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

Doctorat en Sciences / info:eu-repo/semantics/nonPublished

An extracellular bacteriocin-like substance produced by Clostridium acetobutylicum was detected during studies on an industrial fermentation process. The bacteriocin-like substance was not inducible by either ultraviolet light or mitomycin C, and its production was not associated with the induction of a protease. Studies on the mode of action of the bacteriocin-like substance indicated that it had no significant effect on DNA, RNA, or protein synthesis, and it did not cause the loss of intracellular ATP. However, the bacteriocin-like substance was able to lyse SDS-treated cells and cell walls of C. acetobutylicum and was identified as an autolysin. Some of the characteristics of this extracellular autolysin were determined, and after purification it was shown to be a glycoprotein with a molecular weight of 28 000.

It is generally known that media containing simple sugars (sucrose, glucose) and organic nitrogen sources (ammonium) when buffered to acidic pH stimulates aflatoxin production in Aspergillus flavus & A. parasiticus while lactose, nitrate and an alkaline pH inhibit aflatoxin biosynthesis. It has been shown that pH of the growth medium is the most important regulatory factor for aflatoxin biosynthesis since media containing stimulatory carbon and/or nitrogen sources (sucrose and ammonia) do not enhance aflatoxin (or sterigmatocystin) production at alkaline pH. RNA interference (in A. flavus) of the pH regulatory transcription factor, PacC, resulted in aflatoxin production under acidic and alkaline pH conditions whilst wildtype Aspergillus flavus produced aflatoxins only under acidic conditions. This conclusively proved that PacC negatively regulates aflatoxin production at alkaline pH in A. flavus. However the exact mechanism involved in PacC repression of aflatoxin biosynthesis at alkaline pH still remains unknown. The AflR protein is essential for expression of several genes in the aflatoxin biosynthetic cluster. In the current study, sequence analysis of the aflR promoter indicated the presence of two putative PacC binding sites within the aflR promoter of A. flavus 3357WT located at positions -162 and -487 bp from the start codon. The presence of the PacC binding sites in the aflR promoter indicated a possible link between aflR expression and PacC regulation under alkaline conditions. Thus, in this study, it was hypothesized that at alkaline pH, PacC inhibits aflR expression by binding to one or both of the PacC binding sites within the aflR promoter. This in turn, would result in inhibition of aflatoxin biosynthesis since expression of several aflatoxin biosynthetic pathway genes is dependent on activation by AflR. The aim and objective of this study was to test the validity of this hypothesis i.e. that at alkaline pH PacC binds to one or both of its recognition sites within the aflR promoter thereby inhibiting aflR expression which subsequently would result in inhibition of aflatoxin biosynthesis. This was done by first mutating each individual and then both PacC binding sites in the A. flavus 3357 aflR promoter via Single-Joint PCR (SJ-PCR) and fusing the wildtype and each mutated aflR promoter to the Green Fluorescent Protein (gfp) gene and the trpC terminator to yield a functional expression vector. These constructs were then transformed into A. flavus 3357.5. Positive transformants were confirmed to express GFP by fluorescence microscopy and spectrofluorometry. Quantification of GFP protein levels of the various transformants in this study indicated that PacC negatively regulated aflR promoter activity at alkaline pH. RT-qPCR was performed on positive transformants after growth on SLS medium at acidic and alkaline pH to determine if PacC negatively regulated aflR promoter activity at alkaline pH and to determine whether PacC binds preferentially to one or both recognition sites within the aflR promoter. RT-qPCR analysis suggest that PacC binds non-preferentially to both recognition sites within the aflR promoter on sucrose and lactose media at alkaline pH, although mutation of PacC binding site 2 results in a slightly higher expression compared to mutation of PacC binding site 1. Increasing the concentration of an aflatoxin conducive nitrogen source stimulated aflR promoter activity but this was not sufficient to overcome negative regulation by PacC. It is generally known that repression of aflR expression results in repression of aflatoxin biosynthesis irrespective of pH. The results of this study strongly suggest that PacC negatively regulates aflR promoter activity at alkaline pH by binding to one or both PacC recognition sites within the aflR promoter. Since aflR promoter activity is repressed by PacC at alkaline pH, this substantiates the hypothesis that PacC represses aflatoxin biosynthesis by inhibiting expression of aflR. Furthermore, the results of this study indicated that there may be some PacC protein present in the active form at acidic pH irrespective of the carbon source and nitrogen source used in the growth medium. RT-qPCR analysis indicated that any active PacC present at acidic pH may cause repression of the aflR promoter based on the position of the PacC binding site relative to the aflR start codon, although it appears that PacC may have a higher affinity for PacC binding site 2 (which is closer to the aflR start codon).

Orientador: Everardo Magalhães Carneiro / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T11:51:38Z (GMT). No. of bitstreams: 1 Russo_MorganaRejaneRabeloRosa_D.pdf: 4218123 bytes, checksum: b0799db6946f29ef482adbc56a8add73 (MD5) Previous issue date: 2011 / Resumo: O aminoácido leucina é conhecido por ativar a via de síntese protéica muscular. Pesquisas recentes relatam alterações moleculares de proteínas envolvidas na via de síntese protéica após sessões agudas de exercício. No entanto, o efeito da leucina associada ao treinamento aeróbio foi pouco investigado. Sendo assim, o presente estudo teve como objetivo avaliar o papel da suplementação crônica com leucina, durante um programa de exercício aeróbio, sobre proteínas sinalizadoras da via de sensibilidade à insulina e síntese protéica em músculo esquelético e fígado de camundongos adultos. Os animais foram divididos em 4 grupos: controle (C), controle suplementado com leucina (CS), grupo de natação treinado (T) e grupo de natação treinado suplementado com leucina (TS). Após 12 semanas de exercício (1h30, 5 vezes/semana), o peso corporal do grupo T estava menor em relação ao outros grupos. O conteúdo de glicogênio muscular e hepático aumentou nos grupos CS, T e TS comparados ao grupo C. Os animais treinados (T e TS) apresentaram atividade da Cis e sensibilidade à insulina aumentada e menor porcentagem de gordura comparado aos animais controle (C e CS). A suplementação aumentou acentuadamente o teor de insulina e leucina plasmática no CS e TS comparado ao C e T. Em músculo esquelético, a suplementação com leucina, apesar de aumentar a fosforilação do IR, não alterou o contéudo total de IR e IRS-1 e a fosforilação do IRS-1, no entanto, a expressão gênica, o conteúdo total da mTOR e a fosforilação da p70s6k estavam aumentados. O treinamento aeróbio elevou o conteúdo total do IR e a fosforilação do IR e IRS-1, entretanto, reduziu o contéudo da p70s6k e não alterou o contéudo total da mTOR e fosforilação da p70s6k. A associação dos tratamentos elevou o conteúdo total e a fosforilação do IR acima dos valores encontrados quando os tratamentos foram administrados de forma individual e a fosforilação do IRS1 em relação aos grupos controle. Houve elevação da expressão gênica e conteúdo total da mTOR e conteúdo total e fosforilação da p70s6k acima dos valores encontrados para o grupo T, porém abaixo dos valores do grupo CS. No músculo esquelético, os resultados da associação dos tratamentos sugerem que a leucina diminuiu o efeito do exercício aeróbio sobre a via da mTOR/p70s6k. No fígado, a suplementação com leucina aumentou o conteúdo de IR e IRS-1 e a fosforilação do IR, assim como o conteúdo total da mTOR e fosforilação da p70s6k. O treinamento aeróbio, apesar de ter aumentado a fosforilação do IR, diminuiu o conteúdo total de IR, IRS-1, mTOR e p70s6k, assim como, a fosforilação do IRS-1. Quando os tratamentos foram associados, a leucina reverteu o efeito atenuador do exercício sobre a via da mTOR/p70s6k e sobre o IR e IRS-1. Estes resultados indicam que a administração de suplementação crônica com leucina para camundongos adultos durante um programa de exercício aeróbio pode ser importante por proporcionar maior ativação de proteínas da via de síntese protéica em músculo esquelético e fígado quando comparadas à atividade expressa somente em função do exercício aeróbio / Abstract: The amino acid leucine is known to activate the pathway of muscle protein synthesis. Recent surveys have reported molecular alterations after acute exercise sections. However, the effect of leucine associated with the aerobic training was seldom investigated. Thus, this study aimed to evaluate the role of chronic leucine supplementation during a program of aerobic exercise on protein signaling pathway in insulin sensitivity and protein synthesis in skeletal muscle and liver of adult mice. The animals were divided into four groups: control (C), control supplemented with leucine (CS), swim trained group (T) and swim-trained group supplemented with leucine (TS). After 12 weeks of exercise (1h30, 5 times / week), body weight of group T was lower than in the other groups. The glycogen content in muscle and liver increased in T and TS compared with group C. Trained animals (T and TS) had increased activity of Cis, insulin sensitivity and lower fat percentage compared to control animals (C and CS). The supplementation increased significantly the level of plasma insulin and leucine in CS and TS compared to C and T. In skeletal muscle, supplementation with leucine, despite increasing the phosphorylation of IR, did not alter the total content of IR and IRS-1 and phosphorylation of IRS-1; however, gene expression, the entire contents of mTOR and phosphorylation of p70s6k were increased. Aerobic training increased the total content of IR and phosphorylation of IR and IRS-1, however, reduced the content of p70s6k and did not alter the content of total mTOR and phosphorylation of p70s6k. The combination treatment increased the total content and phosphorylation of IR above the values found when the treatments were administered individually and phosphorylation of IRS-1 above the values control groups. There was an elevation of mRNA expression and mTOR total content, and phosphorylation of p70s6k above the values found for the T group, but below the CS group. In skeletal muscle, the results of the association, suggest that leucine reversed the effect of aerobic exercise on the pathway of mTOR/p70s6k. In the liver, leucine supplementation increased the content of IR and IRS-1 and phosphorylation of IR, the entire contents of phosphorylation of mTOR and p70s6k. The aerobic training, although it increased the phosphorylation of IR, decreased the total content of IR, IRS-1, mTOR and p70s6k, as well as the phosphorylation of IRS-1. When the treatments were associated leucine, it reversed the negative effect of exercise on the path of mTOR/p70s6k and the IR and IRS-1. These results indicate that chronic administration of leucine supplementation to adult mice during an aerobic exercise program may be important for making proteins pathways more active to protein synthesis in skeletal muscle and liver when compared to the activity expressed only in terms of aerobic exercise / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Quiescence, a transitory period of non-growth, is a ubiquitous aspect that is present in all organisms. In addition to being present in all forms of life, quiescence is a feature that has been observed in cells that are important for human health, including stem cells in mammals and antibiotic tolerant cells in bacteria. In bacteria, quiescence per se has recently been suggested to underlie the transient tolerance to a wide range of antibiotics. Furthermore, most microbial life exists in a quiescent state. Despite their prevalence and importance, relatively little is known about the physiology of quiescent bacteria. One aspect of bacterial quiescence that has been repeatedly observed is their lowered metabolic activity compared to actively growing bacteria. How do cells that grow and divide enter into a temporary state of non-growth? In particular, how are the energy-intensive processes that are required for growing cells regulated during a non-growing state? The main subject of this thesis is to investigate how protein synthesis, the most energy-intensive process in growing bacterial cells, is regulated during entry into a quiescent phenotype (stationary phase). I first investigate how protein synthesis is regulated using a single cell method that fluorescently tags nascent polypeptide chains. In chapter 3, I show that during entry into stationary phase, protein synthesis is downregulated heterogeneously with one group of cells having comparatively low protein synthesis, resulting in a population that is approximately bimodal. I further show that this bimodality is dependent on a signaling system (PrkC and its partner phosphatase PrpC) that senses cell wall metabolism. I connect signaling from this system to the expression of an enzyme (SasA) that produces a group of nucleotides that are major regulators of growth in bacteria ((pp)pGpp). Lastly, I show that the bimodality is dependent on the three enzymes that synthesize (pp)pGpp. In chapter 4, I explore in detail how the bimodality in protein synthesis is generated. This heterogeneity requires the production of (pp)pGpp by three synthases: SasA, SasB, RelA. I first show that these enzymes differentially affect this bimodality: RelA and SasB are necessary to generate the sub-population exhibiting low protein synthesis, whereas SasA is necessary to generate cells exhibiting comparatively higher protein synthesis. The RelA product (pppGpp) allosterically activates SasB, and I find that the SasA product (pGpp) competitively inhibits this activation. I provide in vivo evidence that this antagonistic interaction mediates the observed heterogeneity in protein synthesis. This chapter, therefore, identifies the mechanism underlying the generation of phenotypic heterogeneity in the central physiological process of protein synthesis. In chapter 5, I next turn to understand the biochemical mechanism by which cells with comparatively low levels of protein synthesis down-regulate this process. I first show that ppGpp is sufficient to inhibit protein synthesis in vivo. I then show that ppGpp inhibits protein synthesis by inhibiting translation initiation directly by binding to the essential GTPase, Initiation Factor 2 (IF2). In collaboration with Ruben Gonzalez’s lab, we also show that ppGpp prevents the allosteric activation of IF2. Finally, I demonstrate that the observed attenuation of protein synthesis during the entry into quiescence is a consequence of the direct interaction of (pp)pGpp and IF2.

No description available.

No description available.

An vitro protein synthesizing system using Sephadex G-25 gel filtration for supernatant purification was used to determine the ability of extracts from embryos of dormant and stratified sugar pine seeds to promote protein synthesis. It was found that the ribosomes from the embryos of dormant seeds were more active than those from stratified seeds but they also required a greater quantity of supernatant protein to produce this activity. The supernatant fraction from stratified embryos was more active than that from dormant embryos but there were indications that this increase was due to seed imbibition. Concentrations of other essential components of the system were the same for both dormant and stratified embryo systems. In each 0.5 m1 sample, maximum incorporation occurred using 0.03-0.06 mg ribosomal protein, 0.795 μmo1es ATP, 1.8 μmo1es PEP, 13.2 μgms pyruvate kinase, 0.090 μmoles GTP, and 0.0375 mg polyuridylic acid. Optimum incubation conditions were at 37°C for 60 minutes. Ribonuclease and protease inhibited phenylalanine incorporation. Ribonuclease activity in the supernatant fraction increased with purification and was significantly higher in the dormant embryos than in the stratified. Protease activity decreased with purification of the supernatant and there was no significant difference between activity in the dormant and stratified embryo supernatant fractions. Protease activity was high in the ribosomal fraction. Ribosomes from dormant embryos appeared to bind more polyuridylic acid than did those from stratified embryos. Resedimented ribosomes consisted primarily of monoribosomes but some polyribosomes were present in both dormant and stratified embryos. Analysis of incubation mixtures produced similar results. The majority of the labelled polyphenylalanine was associated with the monoribosomes. / Master of Science

In the present work, the chromatographic behavior and partial characterization of 5 protein phosphatases, 6 paranitrophenyl phosphate (pnpp) phosphatases and 2 protein kinases from <i>Dictyostelium</i> are described. .Two of the protein phosphatases are shown to have subunit structures. All of the protein phosphatase activities described were able to dephosphorylate sites on proteins that had previously been phosphorylated by the cAMP-dependent protein kinase. Therefore it may be that these phosphatases function <i>in vivo</i> to antagonize the action of the cAMP-dependent protein kinase. Various pnpp phosphatase activities, varying in size, cation requirements and pH optima are described. Since many pnpp phosphatases also function with protein substrates it is possible that these activities represent protein phosphatases that function with phosphoproteins that have not been tested. Evidence for the existence of protein kinase C in Dictyostelium is presented. A histone protein kinase was found in <i>Dictyostelium</i> extracts that eluted from DE52 cellulose at the same salt concentration that rabbit brain protein kinase C did. In addition in one experiment clear dependency on calcium and phospholipids is shown. The purification to homogeneity, and characterization of a low molecular weight autophosphorylating protein kinase, pp20, is described. This protein kinase is a major phosphorylated protein and in addition represents at least 0.03% of the total cellular protein. The autophosphorylation reaction can be inhibited by EDTA but not by EGTA. Following inhibition by EDTA, autophosphorylation can be reactivated by the addition of Mn²⁺, Mg²⁺, or Ca²⁺. Western blotting evidence is presented that shows cross reactivity of pp20 and an 85 KDa protein that may possess casein kinase activity. / Ph. D. / incomplete_metadata