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1.
FEMS Microbiol Lett ; 344(1): 10-7, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23551202

RESUMO

Proteorhodopsins (PRs), light-driven proton pumps, constitute the largest family of the microbial rhodopsins. PRs are widely distributed in the oceanic environment and freshwater, but no bacteria with PRs have been isolated from freshwater so far. To facilitate isolation of the bacteria with PR genes, we constructed a vector system that can be used to clone potential PR genes and render color changes when overexpressed in Escherichia coli. Using this method, we successfully isolated a strain with PR gene from freshwater and identified it as Exiguobacterium sp. JL-3. The full length PR gene was then cloned using the SEFA PCR method. Protein sequence alignment showed that JL-3_PR shares high sequence identity (84-89%) with the PRs from Exiguobacterium strains, but low sequence identity (< 38%) with other PRs. Surprisingly, we could not detect any proton-pumping activity in the native JL-3 cells and protoplasts, but the recombinant JL-3_PR do pump protons when overexpressed in E. coli. Sequence analysis further revealed that the PRs from Exiguobacterium had an unusual lysine as the proton donor instead of the typical acidic residue. These data suggest that JL-3_PR is a sensory PR rather than a proton pump.


Assuntos
Água Doce/microbiologia , Rodopsina/metabolismo , Rodopsinas Microbianas/metabolismo , Sequência de Aminoácidos , Ordem dos Genes , Vetores Genéticos/genética , Dados de Sequência Molecular , Mutação , Filogenia , Prótons , RNA Ribossômico 16S , Rodopsina/genética , Rodopsinas Microbianas/classificação , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de DNA
2.
Cytokine ; 57(1): 37-45, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22088600

RESUMO

CoPoIFN-α is a recombinant non-naturally occurring porcine interferon-α (IFN-α). It was designed by scanning 17 porcine IFN-α nonallelic subtypes and assigning the most frequently occurring amino acid in each position. We used a porcine IFN-α (PoIFN-α) derived from domestic pig as a control. Both porcine IFN-α genes were introduced into yeast expression vector PpICZα-A and expressed in Pichia pastoris. The antiviral unit of these two IFN-αs were assayed in MDBK, PK-15 and MARC-145 cells against vesicular stomatitis virus (VSV), and their inhibitory abilities on pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) replication were also examined, respectively. We found the antiviral activity (units/mg) of CoPoIFN-α was 46.4, 63.6 and 53.5-fold higher than that of PoIFN-α for VSV inhibition in MDBK, PK-15 and MARC-145 cells, 4.8-fold higher for PRV inhibition in PK-15 cells, and 5-fold higher for PRRSV inhibition in MARC-145 cells. Our results also showed that the PRV and PRRSV-specific cytopathic effect (CPE) could be inhibited in the cells pretreated with CoPoIFN-α and PoIFN-α, and the virus titers in the cells pretreated with CoPoIFN-α were lower than those cells pretreated with PoIFN-α by 10-20-fold. The antiproliferative activity of CoPoIFN-α was significantly higher than that of PoIFN-α on a molar basis. The mRNA level of Mx1 and OAS1 genes in PK-15 cells induced by CoPoIFN-α were enhanced about 4.6-fold and 3.2-fold compared to that induced by PoIFN-α. Based on a homology model of CoPoIFN-α and IFNAR2, all of the different residues between native PoIFN-α and CoPoIFN-α were not involved in IFNAR1 binding site, and there is no direct interaction between these residues and IFNAR2, either. We speculate that the higher activity of CoPoIFN-α was likely due to the electrostatic potential introduced by residue Arg156 around the binding site or a structural perturbation caused by these different residues. This may enhance the overall binding affinity of CoPoIIFN-α and the receptors. Thus, CoPoIFN-α may have the potential to be used in therapy of porcine diseases.


Assuntos
Sequência Consenso , Interferon-alfa/biossíntese , Interferon-alfa/genética , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Antivirais/farmacologia , Western Blotting , Bovinos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Efeito Citopatogênico Viral/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Herpesvirus Suídeo 1/efeitos dos fármacos , Interferon-alfa/química , Interferon-alfa/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Pichia , Plasmídeos/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Homologia Estrutural de Proteína , Sus scrofa , Regulação para Cima/efeitos dos fármacos , Vesiculovirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
3.
J Mol Biol ; 367(2): 547-57, 2007 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-17270211

RESUMO

Oxaloacetate decarboxylase is a membrane-bound multiprotein complex that couples oxaloacetate decarboxylation to sodium ion transport across the membrane. The initial reaction catalyzed by this enzyme machinery is the carboxyl transfer from oxaloacetate to the prosthetic biotin group. The crystal structure of the carboxyltransferase at 1.7 A resolution shows a dimer of alpha(8)beta(8) barrels with an active site metal ion, identified spectroscopically as Zn(2+), at the bottom of a deep cleft. The enzyme is completely inactivated by specific mutagenesis of Asp17, His207 and His209, which serve as ligands for the Zn(2+) metal ion, or by Lys178 near the active site, suggesting that Zn(2+) as well as Lys178 are essential for the catalysis. In the present structure this lysine residue is hydrogen-bonded to Cys148. A potential role of Lys178 as initial acceptor of the carboxyl group from oxaloacetate is discussed.


Assuntos
Carboxiliases/química , Carboxil e Carbamoil Transferases/química , Modelos Moleculares , ATPase Trocadora de Sódio-Potássio/química , Vibrio cholerae/enzimologia , Zinco/química , Sequência de Aminoácidos , Sítios de Ligação , Carboxiliases/genética , Carboxil e Carbamoil Transferases/genética , Cátions Bivalentes , Cristalografia por Raios X , Dimerização , Ligação de Hidrogênio , Dados de Sequência Molecular , Mutação , ATPase Trocadora de Sódio-Potássio/genética
4.
J Environ Sci (China) ; 16(2): 332-8, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15137665

RESUMO

Urban human thermal comfort (UHTC) is affected for interacting of weather condition and underlying surface framework of urban area. Urban underlying surface temperature value and Normalized Difference Vegetation Index (NDVI) were calculated using image interpreting and supervised classification technique by ERDAS IMAGE software using 1991 and 1999 Landsat TM images data. Reference to the relational standard of assessing human thermal comfort and other meteorology data of Hangzhou City in summer, air temperature and relative humidity variation of different land types of underlying surface were inversed. By choosing discomfort index as an indictor, the spatial distribution characteristic and the spatial variation degree of UHTC were estimated and mapped on a middle scale, that is, in six districts of Hangzhou. The main characteristics of UHTC spatial variation from 1991 to 1999 were revealed using a GIS-based calculation model. The variation mechanism were analyzed and discussed from the viewpoint of city planning, construction and environmental protection.


Assuntos
Clima , Monitoramento Ambiental/métodos , Temperatura Alta , Saúde da População Urbana , População Urbana , China , Planejamento de Cidades , Planejamento Ambiental , Sistemas de Informação Geográfica , Umidade , Temperatura
5.
J Biol Chem ; 278(36): 33985-91, 2003 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-12821661

RESUMO

A second group of proteorhodopsin-encoding genes (blue-absorbing proteorhodopsin, BPR) differing by 20-30% in predicted primary structure from the first-discovered green-absorbing (GPR) group has been detected in picoplankton from Hawaiian deep sea water. Here we compare BPR and GPR absorption spectra, photochemical reactions, and proton transport activity. The photochemical reaction cycle of Hawaiian deep ocean BPR in cells is 10-fold slower than that of GPR with very low accumulation of a deprotonated Schiff base intermediate in cells and exhibits mechanistic differences, some of which are due to its glutamine residue rather than leucine at position 105. In contrast to GPR and other characterized microbial rhodopsins, spectral titrations of BPR indicate that a second titratable group, in addition to the retinylidene Schiff base counterion Asp-97, modulates the absorption spectrum near neutral pH. Mutant analysis confirms that Asp-97 and Glu-108 are proton acceptor and proton donor, respectively, in retinylidene Schiff base proton transfer reactions during the BPR photocycle as previously shown for GPR, but BPR contains an alternative acceptor evident in its D97N mutant, possibly the same as the second titratable group modulating the absorption spectrum. BPR, similar to GPR, carries out outward light-driven proton transport in Escherichia coli vesicles but with a reduced translocation rate attributable to its slower photocycle. In energized E. coli cells at physiological pH, the net effect of BPR photocycling is to generate proton currents dominated by a triggered proton influx, rather than efflux as observed with GPR-containing cells. Reversal of the proton current with the K+-ionophore valinomycin supports that the influx is because of voltage-gated channels in the E. coli cell membrane. These observations demonstrate diversity in photochemistry and mechanism among proteorhodopsins. Calculations of photon fluence rates at different ocean depths show that the difference in photocycle rates between GPR and BPR as well as their different absorption maxima may be explained as an adaptation to the different light intensities available in their respective marine environments. Finally, the results raise the possibility of regulatory (i.e. sensory) rather than energy harvesting functions of some members of the proteorhodopsin family.


Assuntos
Rodopsina/química , Escherichia coli/metabolismo , Glutamina/química , Concentração de Íons de Hidrogênio , Ionóforos/farmacologia , Leucina/química , Luz , Mutagênese Sítio-Dirigida , Mutação , Potássio/metabolismo , Prótons , Retina/metabolismo , Rodopsina/genética , Rodopsinas Microbianas , Água do Mar , Espectrofotometria , Fatores de Tempo
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