No description available.

No description available.

Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1), demonstrates the need for a much improved, more sophisticated influenza vaccine. Our group has developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC). Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Here we report methodologies for the construction of membrane-bound cytokine fusion constructs in which our cytokine of interest (mouse GM-CSF, mouse IL-2, mouse IL-4) was fused to the membrane anchoring regions of viral Hemagglutinin (HA). Progeny virions, produced from influenza infected MDCK cells expressing membrane-bound cytokines, readily incorporated membrane-bound cytokines during budding and these cytokines on the virus particles retained bioactivity following viral inactivation. In vivo vaccination studies in mice showed enhanced antibody titers and improved protection following lethal challenge in those mice vaccinated with IL-2 and IL-4-bearing CYT-IVAC's compared to the conventional wild-type vaccine without membrane-bound cytokines. In addition, the immune response induced by IL-2 and IL-4-bearing CYT-IVACs was skewed toward Th1 (cellular) mediated immunity compared to the Th2 (humoral) dominated response induced with wild-type vaccination. Cellular mediated immunity afforded by IL-2 and IL-4 CYT-IVACs was manifested as enhanced influenza specific T cell proliferation and activation. In conclusion, we have developed a novel methodology to introduce bioactive membrane-bound cytokines directly into virus particles in order to augment the immunogenicity of inactivated, whole virus influenza vaccines. / Ph. D.

Kyoto University (京都大学) / 0048 / 新制・論文博士 / 博士(工学) / 乙第12200号 / 論工博第3989号 / 新制||工||1438(附属図書館) / 26272 / UT51-2008-C970 / (主査)教授 今中 忠行, 教授 青山 安宏, 教授 森 泰生 / 学位規則第4条第2項該当

ATP is the main cellular energy generated by the enzyme ATP synthase in almost all organisms from bacteria to vertebrates. While malfunction of the ATP synthase complex is responsible for several disease conditions, the enzyme itself can be used as a potent molecular drug target to combat many diseases including microbial infections, cancer, tuberculosis, and obesity. Recent widespread escalation of antibiotic resistant microbes in general and E. coli in particular demands novel alternative approaches to combat microbial infections. Inhibition of ATP synthase by inhibitors such as peptides is known to deprive microbes of required energy, resulting in microbial cell death. Therefore, we have examined the venom peptide induced inhibition of E. coli ATP synthase. It was found that venom peptides completely inhibited E. coli ATP synthase and the process of inhibition was found to be fully reversible. This study also links the antimicrobial properties of peptides in part to the inhibition of ATP synthase. Thus, selective use of ATP synthase as a molecular drug may have an important impact on biology and medicine.

O aproveitamento da energia luminosa por bactérias que produzem hidrogenases poderia aumentar a eficiência do processo de produção de biohidrogênio. Neste trabalho, foi realizada a clonagem do gene que codifica a proteorrodopsina (PR) do isolado metagenômico SAR86 num plasmídeo de expressão para bactérias Gram-negativas. PR é uma proteína ligada ao cromóforo retinal, que, sob iluminação, promove o efluxo de prótons através da membrana celular. O excesso de prótons na face externa da membrana pode servir como substrato para a hidrogenase, resultando em maior eficiência na produção de hidrogênio (2H+ + 2e→ H2). O plasmídeo contendo o gene da PR foi utilizado na transformação genética das bactérias Cupriavidus necator e Escherichia coli, que produzem diversas hidrogenases. Enquanto a PR não se mostrou funcional em C. necator, na linhagem recombinante de E. coli, cultivada em presença de luz e retinal, foi obtido um aumento de até 2,17 vezes na produção de H2 em relação ao cultivo no escuro, desde que a linhagem estivesse produzindo a hidrogenase endógena HYD-4. / The utilization of light energy by hydrogenase producing bacteria could increase the efficiency of the biohydrogen production process. In the present work, the gene coding for proteorhodopsin (PR) of the SAR86 metagenomic lineage was cloned in an expression plasmid for Gram-negative bacteria. PR is an apoprotein linked to the chromophore retinal, which, upon illumination, promotes proton efflux across the cell membrane. The excess of protons on the plasma membrane surface may serve as a substrate for hydrogenases, resulting in a higher efficiency of hydrogen production (2H+ + 2e→ H2). The plasmid containing the PR gene was used to transform the Gram-negative bacteria Cupriavidus necator and Escherichia coli which produce several hydrogenases. Whereas PR did not display functionality in C. necator, in the recombinant E. coli cells, grown under illumination in the presence of retinal, an enhancement up to 2.17 fold in H2 production was found, relative to cells grown under darkness, provided that the cells were expressing the endogenous HYD-4 hydrogenase.

No description available.

No description available.

O aproveitamento da energia luminosa por bactérias que produzem hidrogenases poderia aumentar a eficiência do processo de produção de biohidrogênio. Neste trabalho, foi realizada a clonagem do gene que codifica a proteorrodopsina (PR) do isolado metagenômico SAR86 num plasmídeo de expressão para bactérias Gram-negativas. PR é uma proteína ligada ao cromóforo retinal, que, sob iluminação, promove o efluxo de prótons através da membrana celular. O excesso de prótons na face externa da membrana pode servir como substrato para a hidrogenase, resultando em maior eficiência na produção de hidrogênio (2H+ + 2e→ H2). O plasmídeo contendo o gene da PR foi utilizado na transformação genética das bactérias Cupriavidus necator e Escherichia coli, que produzem diversas hidrogenases. Enquanto a PR não se mostrou funcional em C. necator, na linhagem recombinante de E. coli, cultivada em presença de luz e retinal, foi obtido um aumento de até 2,17 vezes na produção de H2 em relação ao cultivo no escuro, desde que a linhagem estivesse produzindo a hidrogenase endógena HYD-4. / The utilization of light energy by hydrogenase producing bacteria could increase the efficiency of the biohydrogen production process. In the present work, the gene coding for proteorhodopsin (PR) of the SAR86 metagenomic lineage was cloned in an expression plasmid for Gram-negative bacteria. PR is an apoprotein linked to the chromophore retinal, which, upon illumination, promotes proton efflux across the cell membrane. The excess of protons on the plasma membrane surface may serve as a substrate for hydrogenases, resulting in a higher efficiency of hydrogen production (2H+ + 2e→ H2). The plasmid containing the PR gene was used to transform the Gram-negative bacteria Cupriavidus necator and Escherichia coli which produce several hydrogenases. Whereas PR did not display functionality in C. necator, in the recombinant E. coli cells, grown under illumination in the presence of retinal, an enhancement up to 2.17 fold in H2 production was found, relative to cells grown under darkness, provided that the cells were expressing the endogenous HYD-4 hydrogenase.

Infection of barley (Hordeum vulgare) leaves with the rust fungus, Puccinia hordei, causes changes in the host protein synthesis. This thesis analyses these changes in the barley cultivar Triumph following inoculation of 7-day-old leaves with either a virulent or an avirulent race of P. hordei. The initial approach was to isolate membrane-bound polysomes from infected leaves, translate them in vitro and analyse the translation products. These products include the integral membrane proteins which were expected to be involved in the response of the host to the pathogen. A method based on differential centrifugation in the presence of a ribonuclease-inhibiting buffer was developed for separating membrane-bound polysomes from the rest of the cytoplasmic polysomes. Membrane-bound polysomes were found to comprise one fifth of the total polysomes in the leaves. Analysis of the translation products of membrane-bound polysomes by SDS-PAGE showed them to be of higher average molecular weight than those from free polysomes. Comparison of polypeptides produced by membrane-bound polysomes from healthy and inoculated plants showed some differences however the low yield of membrane-bound polysomes made it difficult to obtain conclusive results. Thus it was decided to isolate total polysomes by including 1% Triton X-100 in the extraction buffer. Polysomes were extracted from 12 to 72 h after inoculation. Infection caused a decline in yield of polysomes during this period when compared with healthy leaves of the same age. Polysomes isolated 16 h after inoculation with the virulent race were 20% less efficient at translation than polysomes from control leaves. In contrast polysome isolated from leaves inoculated with the avirulent race were 20% more efficient. Analysis of the labelled translation products by SDS-PAGE and fluorography showed relative increases in the synthesis of some proteins by 16 h after inoculation with either race when compared to products from healthy leaves. Protein synthesis in the infected plants was further analysed by in vivo labelling and one- and two-dimensional PAGE. The fluorographs revealed increased synthesis of a group of proteins from 58 to 116 kDa starting 12 h after inoculation with either race of P. hordei; confirming the results from the polysome translations. Two polypeptides with molecular weights of about 66 kDa were found to increase following infection only with the virulent race. By three days after inoculation with either fungal race the most obvious change in protein synthesis was a marked decrease in the synthesis of the two most prominent polypeptides with molecular weights of 15 and 51 kDa which were considered to be the subunits of ribulose bisphosphate carboxylase. The elicitor hypothesis, in attempting to explain cultivar-specific resistance in plants, postulates that resistance is controlled by the interaction of specific fungal elicitors and plant receptors and that this interaction which only occurs between resistant hosts and avirulent pathogens triggers specific gene expression leading to resistance. This hypothesis does not fit the situation in the barley-P. hordei interaction as protein synthesis showed similar changes following infection with either a virulent or an avirulent race.