A convenient method for the preparation of hydroxyl lactams via regioselective reduction of N-alkyl-3-sulfonyl glutarimides is described. The useful building block is applied to synthesize polysubstituted pyridin-2-one, tacamonine and deplancheine.

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Bovine adenovirus (BAdV)-3, a Mastadenovirus was isolated from the healthy and sick cattle (Darbyshire et al., 1965; Zhu et al., 2011). Like other adenoviruses, BAdV-3 replication is characterized by the temporally regulated expression of genes characterized by early, intermediate and late gene expression. Genus-common, non-structural protein 100K is encoded by late region L6 of BAdV-3. The objective of the present study was to characterize the BAdV-3 100K protein and identify cellular and viral proteins interacting with 100K. Although BAdV-3 100K encoded as 850 amino acid polypeptide (Reddy et al., 1998), rabbit antisera raised against peptides representing N-terminus or C-terminus recognized a protein of 130 kDa at 12-24 hrs post infection, and proteins of 130 kDa, 100 kDa, 95 kDa and 15 kDa at 36-48 hrs post infection. The 100K appeared to be localized to the nucleus and cytoplasm of BAdV-3 infected cells. In contrast, 100K localized predominantly to cytoplasm of transfected cells. However, BAdV-3 infection of cells transfected with 100K-EYFP expressing plasmid detected fluorescent protein in nucleus of the cells suggesting that another viral protein may be required for the nuclear localization of 100K. Using yeast two-hybrid and GST pull-down assays, 100K protein was shown to interact with BAdV-3 33K protein. These results were validated using bimolecular fluorescence complementation (BiFC) assay. Although, 100K protein interacts with 33K protein, co-expression of both proteins in transfected cells did not alter the cytoplasmic localization of 100K. Using GST-pull down assay and BiFC assay, 33K interacting region of 100K was localized to a stretch of 13 amino acids (624-637). Repeated attempts were not successful in rescuing a recombinant BAdV-3 expressing mutant 100K (containing deletion of amino acids 624-637). The interaction of cellular protein(s) with 100K was determined by mass spectrometric analysis of immunoprecipitated 100K. Mass spectrometry of immunoprecipitate obtained by immunoprecipitating 100K protein from BAdV-3 infected cells harvested at 48 hrs post infection identified six proteins including dynein light chain (DYNLT)1. The initial identified interaction of 100K with DYNLT1 was confirmed by the yeast two-hybrid assay, co-immunoprecipitation assay and BiFC assays. Furthermore, DYNLT1 interacting domain of 100K protein of BAdV-3 was found to be located between 499-587 amino acids. Co-expression of BAdV-3 100K-EY fusion protein with myc epitope tagged DYNLT1 protein did not alter the localization of 100K-EY fusion protein. The investigation into the differences in the subcellular localization of the 100K protein in the transfected and infected cells lead to identification of the cleavage by adenoviral protease. Subsequent analysis suggested that BAdV-3 protease cleaves 100K at two identified potential protease cleavage sites (amino acid 740-745 and 781-786) in transfected or BAdV-3 infected cells. Although protease encoded by human adenovirus (HAdV)-5 or porcine adenovirus (PAdV)-3 also cleaved BAdV-3 100K at potential identified protease cleavage sites, no such cleavage of 100K encoded by HAdV-5 or PAdV-3 could be detected in cells expressing virus specific protease. Successful isolation of recombinant BAdV-3 expressing mutant protease (substitution of alanine for glycine in potential protease cleavage site) suggested that cleavage of BAdV-3 100K by viral protease is not essential for viral replication. However, further analysis observed less virus in the supernatant of cells infected with mutant BAdV-3 compared to WT BAdV-3 suggesting a possible role for cleaved C-terminal fagment in lysis of infected cells. Co-expression of BAdV-100K with other late viral proteins suggested that the 100K-EYFP fusion protein localized to the nucleus in cells co-expressing BAdV-3 protease-DsRed fusion protein. Interestingly, only C-terminal cleaved fragment of 100K localizes to the nucleus in BAdV-3 protease expressing cells. Further analysis suggested that C-terminal fragment localizing to the nucleus contains a bipartite nuclear localization signal, which is recognized by importin α3. Our results suggest that the N-terminal part of 100K may be retained in the cytoplasm by interaction with Tctex1 (DYNLT1). Our study provides for the first time a plasmid co-transfection system for the study of the protease cleavage of viral proteins. Moreover, this is the first report of cleavage of any non-structural viral proteins by adenoviral protease in infected cells.

No description available.

As an alternative to the Fibonacci heap, and a variation of the 2-3 heap data structure by Tadao Takaoka, this research presents the 3-4 heap data structure. The aim is to prove that the 3-4 heap, like its counter-part 2-3 heap, also supports n insert, n delete-min, and m decrease-key operations, in O(m + nlog n) time. Many performance tests were carried out during this research comparing the 3-4 heap against the 2-3 heap and for a narrow set of circumstances the 3-4 heap outperformed the 2-3 heap. The 2-3 heap has got a structure based on dimensions which are rigid using ternary linking and this path is made up of three nodes linked together to form a trunk, and the trunk is permitted to shrink by one. If further shrinkage is required then an adjustment is made by moving a few nodes from nearby positions to ensure the heaps rigid dimensions are retained. Should this no longer be the case, then the adjustment will trigger a make-up event, which propagates to higher dimensions, and requires amortised analysis. To aid amortised analysis, the trunk is given a measurement value called potential and this is the number of comparisons required to place each node into its correct position in ascending order using linear search. The divergence of the 3-4 heap from the 2-3 heap is that the trunk maximum is increased by one to four and is still permitted to shrink by one. This modified data structure will have a wide range of applications as the data storage mechanism used by graph algorithms such as Dijkstra's 'Single Source Shortest Path'.

Techn. Universiẗat, Diss., 2001--Darmstadt.

Th. doct.-ing.--Bordeaux 1, 1977. N°: 260.

BACKGROUND: Acute pancreatitis (AP) is a sterile initiator of systemic inflammation that can trigger multiple organ dysfunction syndrome (MODS). In the acute phase of AP, the kynurenine pathway of tryptophan metabolism plays an important role in the genesis of AP-MODS in experimental animal models, but it is unknown whether the pathway is activated in human AP. Human data are required to support the rationale for kynurenine 3- monooxygenase (KMO) inhibition as a treatment for AP-MODS and reinforce the translational potential. Additionally, as respiratory dysfunction is frequent in severe AP, the role of lung ultrasonography in severity stratification deserves investigation. Furthermore, the effect of AP-MODS on long-term survival is unknown. OBJECTIVES: My objectives were to: 1) Define the temporal and quantitative relationship of kynurenine metabolites with the onset and severity of APMODS, 2) Investigate the value of lung ultrasonography in the early diagnosis of respiratory dysfunction in human AP-MODS, and 3) Examine whether early AP-MODS impacts on long-term survival. METHODS: 1) A prospective, observational, clinical experimental medicine study titled “Inflammation, Metabolism, and Organ Failure in Acute Pancreatitis” (IMOFAP) was performed. For 90 days, consecutive patients with a potential diagnosis of AP were recruited and venous blood was sampled at 0, 3, 6, 12, 24, 48, 72 and 168 hours post-recruitment. Kynurenine metabolite concentrations were measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and analysed in the context of clinical data, disease severity indices, and cytokine profiles. 2) In a nested cohort within IMOFAP, 41 participants underwent lung ultrasonography to evaluate whether this imaging modality can detect respiratory dysfunction in AP. 3) Survival data for a prospectively maintained database of patients with AP was analysed after accounting for in-hospital deaths. RESULTS: 1) During the IMOFAP study, 79 patients were recruited with an elevated serum amylase, of which 57 patients met the diagnostic criteria for AP; 9 had severe disease. Temporal profiling revealed early tryptophan depletion and contemporaneous elevation of plasma concentrations of 3- hydroxykynurenine, which paralleled systemic inflammation and AP severity. 2) Lung ultrasonography findings correlated with respiratory dysfunction. 3) 694 patients were followed up for a median of 8.8 years. AP-MODS conferred a deleterious effect on overall survival which persisted after the exclusion of inhospital deaths (10.0 years, 95% C.I. = 9.4-10.6 years) compared to AP without MODS (11.6 years, 95% C.I. = 11.2-11.9 years; P = 0.001). This effect was independent of age. CONCLUSIONS: In the acute phase of AP, metabolic flux through KMO is elevated and proportionate to AP severity. Lung ultrasonography may be a useful technique for evaluating AP-MODS. AP-MODS is an independent predictor of long-term mortality. Together, this work reinforces the rationale for investigating early phase KMO inhibition as a therapeutic strategy in humans.

Devido ao grande avanço na descoberta de novos fármacos a partir de compostos naturais, tornou-se interessante avaliar o potencial antiproliferativo de moléculas isoladas de extratos de plantas. Este trabalho prioriza o estudo da atividade antitumoral de benzofenonas (carifenona A e carifenona B) e floroglucionóis (japonicina A e uliginosina B), isolados das espécies nativas do sul do Brasil, Hypericum carinatum e Hypericum myrianthum, respectivamente, bem como a associação destes com quimioterápicos utilizados na clínica. Os experimentos propostos foram realizados em modelos in vitro, utilizando diferentes tipos de linhagens tumorais humanas comercialmente disponíveis. Foi avaliado o efeito de diferentes doses destes compostos através de experimentos de viabilidade e sobrevivência celular, análise morfométrica nuclear (NMA) e citometria de fluxo. Na análise estatística foi utilizada a variância de uma via (ANOVA) seguida de teste post-hoc (Tukey). Os resultados foram expressos como média ± erro padrão da média (SEM), sendo valores de P menores do que 0,05 considerados significativos. Verificamos que nas linhagens de adenocarcinoma de ovário e colorretal e de glioblastoma (OVCAR-3, HT-29 e U-251) ocorreu uma diminuição significativa na viabilidade celular quando tratadas com a dose de 100μg/mL tanto de carifenona A como de carifenona B, enquanto os compostos japonicina A (50μg/mL) e uliginosina B (20μg/mL) só foram ativos na linhagem OVCAR-3. Dentre as associações com quimioterápicos, a única que apresentou efeito sinérgico foi a combinação de japonicina A e paclitaxel na linhagem OVCAR-3. A partir deste momento selecionamos a japoncina A para dar continuidade aos estudos. Este composto foi avaliado frente a outros tipos de linhagens tumorais, sendo ativa somente em células de adenocarcinoma de ovário e próstata (OVCAR-3 e PC-3). Na linhagem PC-3, a análise do ciclo celular demonstrou decréscimo da fase G1 e indução ao arraste da fase G2, assim como, através da técnica de NMA, foi verificado um aumento de células apoptóticas, quando as células foram tratadas com japonicina A. Estudos moleculares devem ser realizados para melhor entendimento do mecanismo de ação da japonicina A, composto que pode servir de modelo para o desenho de fármacos mais específicos para este tipo de neoplasia. / Due to the great progress in the discovery of new drugs from natural compounds, it has become interesting to evaluate the antiproliferative activity of molecules isolated from plant extracts. This work emphasizes the study of antitumor activity of benzophenones (cariphenone A and cariphenone B) and phloroglucionols (japonicin A and uliginosin B), isolated from Hypericum species native to southern Brazil, H. carinatum and H. myrianthum, respectively, as well as their association with chemotherapeutic drugs used in the clinic. The proposed experiments were performed in vitro using commercially available cell lines. The effect of different doses of these compounds were evaluated via cell viability and survival assay, nuclear morphometric analysis (NMA) and flow cytometry. One way analysis of variance (ANOVA) followed by post hoc tests (Tukey) were utilized for statistical analysis. Results were expressed as mean ± standard error of the mean (SEM), and P values less than 0.05 were considered significant. We found that in ovarian, colorectal (adenocarcinoma) and glioblastoma cell lines (OVCAR-3, HT-29 and U-251) a significant decrease in cell viability occurred when these were treated with a dose of 100μg/mL of cariphenone A and B, while compounds japonicin A (50μg/mL) and uliginosin B (20μg/mL) were active only in OVCAR- 3. Among the associations with chemotherapeutic agents, only japonicin A presented a synergistic effect with paclitaxel in the OVCAR-3 cell line. We then selected japonicin A for evaluation against other cell lines, but its effects were only observed in ovary and prostate adenocarcinoma cell lines (OVCAR-3 e PC-3). In PC-3, the cell cycle revealed a decreased in the G1 phase and induction of G2 arrest, the NMA showed an increase in apoptotic cells when cells were treated with japonicin A. More studies should be conducted to better understand the mechanisms of action of japonicin A, since this compound may serve as pharmacophore model for the design of more specific drugs to treat this tumor type.

Devido ao grande avanço na descoberta de novos fármacos a partir de compostos naturais, tornou-se interessante avaliar o potencial antiproliferativo de moléculas isoladas de extratos de plantas. Este trabalho prioriza o estudo da atividade antitumoral de benzofenonas (carifenona A e carifenona B) e floroglucionóis (japonicina A e uliginosina B), isolados das espécies nativas do sul do Brasil, Hypericum carinatum e Hypericum myrianthum, respectivamente, bem como a associação destes com quimioterápicos utilizados na clínica. Os experimentos propostos foram realizados em modelos in vitro, utilizando diferentes tipos de linhagens tumorais humanas comercialmente disponíveis. Foi avaliado o efeito de diferentes doses destes compostos através de experimentos de viabilidade e sobrevivência celular, análise morfométrica nuclear (NMA) e citometria de fluxo. Na análise estatística foi utilizada a variância de uma via (ANOVA) seguida de teste post-hoc (Tukey). Os resultados foram expressos como média ± erro padrão da média (SEM), sendo valores de P menores do que 0,05 considerados significativos. Verificamos que nas linhagens de adenocarcinoma de ovário e colorretal e de glioblastoma (OVCAR-3, HT-29 e U-251) ocorreu uma diminuição significativa na viabilidade celular quando tratadas com a dose de 100μg/mL tanto de carifenona A como de carifenona B, enquanto os compostos japonicina A (50μg/mL) e uliginosina B (20μg/mL) só foram ativos na linhagem OVCAR-3. Dentre as associações com quimioterápicos, a única que apresentou efeito sinérgico foi a combinação de japonicina A e paclitaxel na linhagem OVCAR-3. A partir deste momento selecionamos a japoncina A para dar continuidade aos estudos. Este composto foi avaliado frente a outros tipos de linhagens tumorais, sendo ativa somente em células de adenocarcinoma de ovário e próstata (OVCAR-3 e PC-3). Na linhagem PC-3, a análise do ciclo celular demonstrou decréscimo da fase G1 e indução ao arraste da fase G2, assim como, através da técnica de NMA, foi verificado um aumento de células apoptóticas, quando as células foram tratadas com japonicina A. Estudos moleculares devem ser realizados para melhor entendimento do mecanismo de ação da japonicina A, composto que pode servir de modelo para o desenho de fármacos mais específicos para este tipo de neoplasia. / Due to the great progress in the discovery of new drugs from natural compounds, it has become interesting to evaluate the antiproliferative activity of molecules isolated from plant extracts. This work emphasizes the study of antitumor activity of benzophenones (cariphenone A and cariphenone B) and phloroglucionols (japonicin A and uliginosin B), isolated from Hypericum species native to southern Brazil, H. carinatum and H. myrianthum, respectively, as well as their association with chemotherapeutic drugs used in the clinic. The proposed experiments were performed in vitro using commercially available cell lines. The effect of different doses of these compounds were evaluated via cell viability and survival assay, nuclear morphometric analysis (NMA) and flow cytometry. One way analysis of variance (ANOVA) followed by post hoc tests (Tukey) were utilized for statistical analysis. Results were expressed as mean ± standard error of the mean (SEM), and P values less than 0.05 were considered significant. We found that in ovarian, colorectal (adenocarcinoma) and glioblastoma cell lines (OVCAR-3, HT-29 and U-251) a significant decrease in cell viability occurred when these were treated with a dose of 100μg/mL of cariphenone A and B, while compounds japonicin A (50μg/mL) and uliginosin B (20μg/mL) were active only in OVCAR- 3. Among the associations with chemotherapeutic agents, only japonicin A presented a synergistic effect with paclitaxel in the OVCAR-3 cell line. We then selected japonicin A for evaluation against other cell lines, but its effects were only observed in ovary and prostate adenocarcinoma cell lines (OVCAR-3 e PC-3). In PC-3, the cell cycle revealed a decreased in the G1 phase and induction of G2 arrest, the NMA showed an increase in apoptotic cells when cells were treated with japonicin A. More studies should be conducted to better understand the mechanisms of action of japonicin A, since this compound may serve as pharmacophore model for the design of more specific drugs to treat this tumor type.