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New Potential Drugs Targets For Burkholderia: Insights From In Silico Analyses
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[P-M.61] Development of Novel Expression Cassettes for Adrenomedullin Gene Using Genomic DNA/cDNA Hybrids Hyunjin Hwang, SaetByeul Lee, Jonghoe Byun ∗ Department of Molecular Biology, Dankook University, Republic of Korea Keywords: Adrenomedullin; Expression; Peptide; Hybrid Adrenomedullin (AM) is a kind of pluripotent peptide which was originally isolated from pheochromocytoma extracts. It is primarily expressed in adrenal medulla, kidney, heart, aorta, lung, and liver. This peptide has many biological functions such as vasodilatation, regulation of permeability, endothelial apoptosis inhibition and promotion of angiogenesis. The human AM gene is located on Chromosome 11 and composed of 4 exons and 3 introns. Although introns are removed by splicing during the processing to mRNA, their role with respect to gene expression level was poorly studied. To this end, various combinations of exons and introns were made for expression of AM and the corresponding constructs were generated. Genomic DNA, cDNA and the processed form of AM (preproAM) were also constructed. The preproAM is enzymatically processed to yield two C-terminal amidated peptides including AM and pro-AM N-terminal 20 peptide (PAMP). Native introns 1, 2, 3 and their combinations were placed to their original places between exons, thus giving 10 genomic DNA/cDNA hybrids for AM expression. These constructs were cloned into pcDNA3.1 (+) vector, and then transfected in HEK293 cells for comparative analysis of AM mRNA level. Each construct was co-transfected with the nlsLacZ expression vector for normalization of transfection efficiency. AM mRNA level was measured by real-time RT-PCR with sybergreen dye. Interestingly, the highest AM mRNA level was detected in the construct containing intron 2. On the other hand, the constructs having intron 1 showed decreased level of AM mRNA. Further characterization of these constructs and introns are currently underway and preliminary results including nature of the peptides produced will be presented at the meeting. doi:10.1016/j.jbiotec.2010.09.649 [P-M.62] New Potential Drugs Targets For Burkholderia: Insights From In Silico Analyses Elena Perrin 1,∗ , Marco Fondi 1 , Maria Cristiana Papaleo 1 , Isabel Maida 1 , Maria Rosalia Pasca 2 , Silvia Buroni 2 1
University of Florence, Italy University of Pavia, Italy Keywords: Drug targets; Burkholderia; RND; Antibiotics resistance 2
The genus Burkholderia includes a variety of species inhabiting different ecological niches, with opportunistic human pathogenic strains, exhibiting an increasing resistance to antibiotic. In this context a major role is played by multidrug efflux pumps belonging to RND family that allow bacterial cells to extrude a wide range of different substrates, including antibiotics. In this work we performed a deep analysis of the structure and distribution of RND proteins in the 21 Burkholderia completely sequenced genomes in order to study the phylogenetic distribution of RND proteins across the genus Burkholderia. To this purpose a BLAST probing of the 21 Burkholderia genomes using experimentally characterized ceoB sequences (one of the RND family counterpart in the genus
S449
Burkholderia) as seed, allowed the assembly of a dataset comprising 254 putative RND proteins. An extensive phylogenetic analysis revealed the occurrence of several independent events of gene loss and duplication across the different lineages of the genus Burkholderia, leading to notable differences in the number of paralogs between different genomes. A putative substrate [antibiotics (HAE1 proteins)/heavy-metal (HME proteins)] was also assigned to most of these proteins. No correlation was found between neither the ecological niche nor the lifestyle of Burkholderia strains and the number/type of efflux pumps they possessed, while a relation can be found with genome size and taxonomy. Remarkably, we observed that only HAE1 proteins are mainly responsible for the different number of proteins observed in strains of the same species. The complete knowledge of the presence and distribution of RND proteins in Burkholderia species may serve as a basis for future experimental tests, focused especially on HAE1 proteins, aimed at the identification of novel targets in antimicrobial therapy against Burkholderia species. doi:10.1016/j.jbiotec.2010.09.650 [P-M.63] Characteristics of novel tyrosinase inhibitor from Zygomycota Mucor subtilissimus Mita Yukiko ∗ , Takano Maki, Hoshino Kazuhiro University of Toyama, Japan Keywords: Tyrosinase inhibitor; Zygomycota; Mucor subtilissimus; Cosmetics Tyrosinase inhibitor such as arbutin, cysteine etc. was used as main components of whitening cosmetics. However, these compounds have serious problems concerning for melanin synthesis inhibit activity and toxicity for skin cells. Therefore, we paid attention to Zygomycota (mainly, Mucor genus) as a new resource for the production of tyrosinase inhibitor. In this research, we attempted the screening for new tyrosinase inhibitor from the broth of the genus. First, the productions of tyrosinase inhibitors were estimated by measuring the two inhibit activities in the cultures of 82 kinds of fungi. The inhibitory activity of the tyrosinase by sample was evaluated by the monophenolase activity with L-tyrosine and the diphenolase activity with L-DOPA, respectively. As these results, the broth obtained by cultivating M.subtilissimus NBRC 6755 for 72 h with glucose has higher inhibitor activities. In the monophenolase activity, its inhibition value was markedly-increased from 119 s to 294 s. Further, the broth showed the inhibition effect of 56% in a diphenolase activity compared with the control. To purify the inhibitor from the broth, the broth was concentrated, extracted by methanol, and fractionated by HPLC, and then the inhibition material was obtained. The material was showed a mixed type inhibition for diphenolase activity, and the inhibition constant Ki on DOPA was 2.85 g/L. Moreover, it was found the structure was furfural like by a GC-MS assay. Further, the inhibitor also showed the inhibition effect on tyrosinase after heat-treatment for 5 min at 100◦ C. Next, to estimate the ability of the inhibitor when in vivo the inhibition assay for melanin synthesis and the toxicity assay were performed by using mouse B16 melanoma cell, the amount of melanin synthesis decreased up to 43% at the inhibitor concentration of 10 mg/L and further the toxicity was not admitted. doi:10.1016/j.jbiotec.2010.09.651
[P-M.61] Development of Novel Expression Cassettes for Adrenomedullin Gene Using Genomic DNA/cDNA Hybrids Hyunjin Hwang, SaetByeul Lee, Jonghoe Byun ∗ Department of Molecular Biology, Dankook University, Republic of Korea Keywords: Adrenomedullin; Expression; Peptide; Hybrid Adrenomedullin (AM) is a kind of pluripotent peptide which was originally isolated from pheochromocytoma extracts. It is primarily expressed in adrenal medulla, kidney, heart, aorta, lung, and liver. This peptide has many biological functions such as vasodilatation, regulation of permeability, endothelial apoptosis inhibition and promotion of angiogenesis. The human AM gene is located on Chromosome 11 and composed of 4 exons and 3 introns. Although introns are removed by splicing during the processing to mRNA, their role with respect to gene expression level was poorly studied. To this end, various combinations of exons and introns were made for expression of AM and the corresponding constructs were generated. Genomic DNA, cDNA and the processed form of AM (preproAM) were also constructed. The preproAM is enzymatically processed to yield two C-terminal amidated peptides including AM and pro-AM N-terminal 20 peptide (PAMP). Native introns 1, 2, 3 and their combinations were placed to their original places between exons, thus giving 10 genomic DNA/cDNA hybrids for AM expression. These constructs were cloned into pcDNA3.1 (+) vector, and then transfected in HEK293 cells for comparative analysis of AM mRNA level. Each construct was co-transfected with the nlsLacZ expression vector for normalization of transfection efficiency. AM mRNA level was measured by real-time RT-PCR with sybergreen dye. Interestingly, the highest AM mRNA level was detected in the construct containing intron 2. On the other hand, the constructs having intron 1 showed decreased level of AM mRNA. Further characterization of these constructs and introns are currently underway and preliminary results including nature of the peptides produced will be presented at the meeting. doi:10.1016/j.jbiotec.2010.09.649 [P-M.62] New Potential Drugs Targets For Burkholderia: Insights From In Silico Analyses Elena Perrin 1,∗ , Marco Fondi 1 , Maria Cristiana Papaleo 1 , Isabel Maida 1 , Maria Rosalia Pasca 2 , Silvia Buroni 2 1
University of Florence, Italy University of Pavia, Italy Keywords: Drug targets; Burkholderia; RND; Antibiotics resistance 2
The genus Burkholderia includes a variety of species inhabiting different ecological niches, with opportunistic human pathogenic strains, exhibiting an increasing resistance to antibiotic. In this context a major role is played by multidrug efflux pumps belonging to RND family that allow bacterial cells to extrude a wide range of different substrates, including antibiotics. In this work we performed a deep analysis of the structure and distribution of RND proteins in the 21 Burkholderia completely sequenced genomes in order to study the phylogenetic distribution of RND proteins across the genus Burkholderia. To this purpose a BLAST probing of the 21 Burkholderia genomes using experimentally characterized ceoB sequences (one of the RND family counterpart in the genus
S449
Burkholderia) as seed, allowed the assembly of a dataset comprising 254 putative RND proteins. An extensive phylogenetic analysis revealed the occurrence of several independent events of gene loss and duplication across the different lineages of the genus Burkholderia, leading to notable differences in the number of paralogs between different genomes. A putative substrate [antibiotics (HAE1 proteins)/heavy-metal (HME proteins)] was also assigned to most of these proteins. No correlation was found between neither the ecological niche nor the lifestyle of Burkholderia strains and the number/type of efflux pumps they possessed, while a relation can be found with genome size and taxonomy. Remarkably, we observed that only HAE1 proteins are mainly responsible for the different number of proteins observed in strains of the same species. The complete knowledge of the presence and distribution of RND proteins in Burkholderia species may serve as a basis for future experimental tests, focused especially on HAE1 proteins, aimed at the identification of novel targets in antimicrobial therapy against Burkholderia species. doi:10.1016/j.jbiotec.2010.09.650 [P-M.63] Characteristics of novel tyrosinase inhibitor from Zygomycota Mucor subtilissimus Mita Yukiko ∗ , Takano Maki, Hoshino Kazuhiro University of Toyama, Japan Keywords: Tyrosinase inhibitor; Zygomycota; Mucor subtilissimus; Cosmetics Tyrosinase inhibitor such as arbutin, cysteine etc. was used as main components of whitening cosmetics. However, these compounds have serious problems concerning for melanin synthesis inhibit activity and toxicity for skin cells. Therefore, we paid attention to Zygomycota (mainly, Mucor genus) as a new resource for the production of tyrosinase inhibitor. In this research, we attempted the screening for new tyrosinase inhibitor from the broth of the genus. First, the productions of tyrosinase inhibitors were estimated by measuring the two inhibit activities in the cultures of 82 kinds of fungi. The inhibitory activity of the tyrosinase by sample was evaluated by the monophenolase activity with L-tyrosine and the diphenolase activity with L-DOPA, respectively. As these results, the broth obtained by cultivating M.subtilissimus NBRC 6755 for 72 h with glucose has higher inhibitor activities. In the monophenolase activity, its inhibition value was markedly-increased from 119 s to 294 s. Further, the broth showed the inhibition effect of 56% in a diphenolase activity compared with the control. To purify the inhibitor from the broth, the broth was concentrated, extracted by methanol, and fractionated by HPLC, and then the inhibition material was obtained. The material was showed a mixed type inhibition for diphenolase activity, and the inhibition constant Ki on DOPA was 2.85 g/L. Moreover, it was found the structure was furfural like by a GC-MS assay. Further, the inhibitor also showed the inhibition effect on tyrosinase after heat-treatment for 5 min at 100◦ C. Next, to estimate the ability of the inhibitor when in vivo the inhibition assay for melanin synthesis and the toxicity assay were performed by using mouse B16 melanoma cell, the amount of melanin synthesis decreased up to 43% at the inhibitor concentration of 10 mg/L and further the toxicity was not admitted. doi:10.1016/j.jbiotec.2010.09.651