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71.
72.
To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and bromocriptine by FRET from the substrate fluorophore to the heme, and by absorbance spectroscopy. These approaches combined with an innovative setup of titration-by-dilution and continuous variation (Job's titration) experiments allowed us to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or bromocriptine or the type-II spectral changes caused by PMA. The 1-PB-induced spin shift in CYP3A4 reveals prominent homotropic cooperativity, which is characterized by a Hill coefficient of 1.8 +/- 0.3 (S50 = 8.0 +/- 1.1 microM). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting KD values of 0.31 +/- 0.08 microM and 7.1 +/- 2.3 microM, respectively. The binding of all three substrates monitored by FRET in titration-by-dilution experiments at an enzyme:substrate ratio of 1 reveals a simple bimolecular interaction with KD values of 0.16 +/- 0.09, 4.8 +/- 1.4, and 0.18 +/- 0.09 microM for 1-PB, PMA, and bromocriptine, respectively. Correspondingly, Job's titration experiments showed that the 1-PB-induced spin shift reflects the formation of a complex of the enzyme with two substrate molecules, while bromocriptine and PMA exhibit 1:1 binding stoichiometry. Combining the results of Job's titrations with the value of KD obtained in our FRET experiments, we demonstrate that the interactions of CYP3A4 with 1-PB obey a sequential binding mechanism, where the spin transition is triggered by the binding of 1-PB to the low-affinity site, which becomes possible only upon saturation of the high-affinity site. 相似文献
73.
Tato I Matilla I Arechaga I Zunzunegui S de la Cruz F Cabezon E 《The Journal of biological chemistry》2007,282(35):25569-25576
Conjugative systems contain an essential integral membrane protein involved in DNA transport called the Type IV coupling protein (T4CP). The T4CP of conjugative plasmid R388 is TrwB, a DNA-dependent ATPase. Biochemical and structural data suggest that TrwB uses energy released from ATP hydrolysis to pump DNA through its central channel by a mechanism similar to that used by F1-ATPase or ring helicases. For DNA transport, TrwB couples the relaxosome (a DNA-protein complex) to the secretion channel. In this work we show that TrwA, a tetrameric oriT DNA-binding protein and a component of the R388 relaxosome, stimulates TrwBDeltaN70 ATPase activity, revealing a specific interaction between the two proteins. This interaction occurs via the TrwA C-terminal domain. A 68-kDa complex between TrwBDeltaN70 and TrwA C-terminal domain was observed by gel filtration chromatography, consistent with a 1:1 stoichiometry. Additionally, electron microscopy revealed the formation of oligomeric TrwB complexes in the presence, but not in the absence, of TrwA protein. TrwBDeltaN70 ATPase activity in the presence of TrwA was further enhanced by DNA. Interestingly, maximal ATPase rates were achieved with TrwA and different types of dsDNA substrates. This is consistent with a role of TrwA in facilitating the interaction between TrwB and DNA. Our findings provide a new insight into the mechanism by which TrwB recruits the relaxosome for DNA transport. The process resembles the mechanism used by other DNA-dependent molecular motors, such as the RuvA/RuvB system, to be targeted to the DNA followed by hexamer assembly. 相似文献
74.
Mapping and characterization of the primary and anamnestic H-2(d)-restricted cytotoxic T-lymphocyte response in mice against human metapneumovirus 下载免费PDF全文
Melendi GA Zavala F Buchholz UJ Boivin G Collins PL Kleeberger SR Polack FP 《Journal of virology》2007,81(20):11461-11467
Cytotoxic T lymphocytes (CTLs) are important for the control of virus replication during respiratory infections. For human metapneumovirus (hMPV), an H-2(d)-restricted CTL epitope in the M2-2 protein has been described. In this study, we screened the hMPV F, G, N, M, M2-1, and M2-2 proteins using three independent algorithms to predict H-2(d) CTL epitopes in BALB/c mice. A dominant epitope (GYIDDNQSI) in positions 81 to 89 of the antitermination factor M2-1 and a subdominant epitope (SPKAGLLSL) in N(307-315) were detected during the anti-hMPV CTL response. Passive transfer of CD8(+) T-cell lines against M2-1(81-89) and N(307-315) protected Rag1(-/-) mice against hMPV challenge. Interestingly, diversification of CTL targets to include multiple epitopes was observed after repetitive infections. A subdominant response against the previously described M2-2 epitope was detected after the third infection. An understanding of the CTL response against hMPV is important for developing preventive and therapeutic strategies against the virus. 相似文献
75.
Mutlu MB Martínez-García M Santos F Peña A Guven K Antón J 《FEMS microbiology ecology》2008,65(3):474-483
Tuz Lake is an inland thalassohaline water body located in central Anatolia that contributes to 60% of the total salt production in Turkey per year. The microbiota inhabiting this lake has been studied by FISH, denaturing gradient gel electrophoresis of PCR-amplified fragments of 16S rRNA genes, and 16S rRNA gene clone library analysis. Total cell counts per milliliter (1.38 × 107 ) were in the range of the values normally found for hypersaline environments. The proportion of Bacteria to Archaea in the community detectable by FISH was one to three. 16S rRNA gene clone libraries indicated that the archaeal assemblage was dominated by members of the Square Haloarchaea of the Walsby group, although some other groups were also found. Bacteria were dominated by members of the Bacteroidetes , including Salinibacter ruber -related phylotypes. Because members of Bacteroidetes are widely present in different hypersaline environments, a phylogenetic analysis of 16S rRNA gene sequences from Bacteroidetes retrieved from these environments was carried out in order to ascertain whether they formed a unique cluster. Sequences retrieved from Tuz Lake and a group of sequences from other hypersaline environments clustered together in a branch that could be considered as the 'halophilic branch' within the Bacteroidetes phylum. 相似文献
76.
Maciel-de-Freitas R Peres RC Alves F Brandolini MB 《Memórias do Instituto Oswaldo Cruz》2008,103(6):602-605
In this report, the efficiency of Adultrap under field conditions is compared to a CDC backpack aspirator and to MosquiTRAP. An urban dengue-endemic area of Rio de Janeiro was selected to evaluate the efficiency of mosquito traps in capturing Aedes aegypti females. Adultrap and aspirator captured similar numbers of Ae. aegypti females, with the former showing high specificity to gravid individuals (93.6%). A subsequent mark-release-recapture experiment was conducted to evaluate Adultrap and MosquiTRAP efficiency concomitantly. With a 6.34% recapture rate, MosquiTRAP captured a higher mean number of female Ae. aegypti per trap than Adultrap (chi2 = 14.26; df = 1; p < 0,05). However, some MosquiTRAPs (28.12%) contained immature Ae. aegypti after 18 days of exposure in the field and could be pointed as an oviposition site for female mosquitoes. Both trapping methods, designed to collect gravid Ae. aegypti females, seem to be efficient, reliable and may aid routine Ae. aegypti surveillance. 相似文献
77.
Bartacek J Fermoso FG Baldó-Urrutia AM van Hullebusch ED Lens PN 《Journal of industrial microbiology & biotechnology》2008,35(11):1465-1474
The influence of cobalt speciation on the toxicity of cobalt to methylotrophic methanogenesis in anaerobic granular sludge
was investigated. The cobalt speciation was studied with three different media that contained varying concentrations of complexing
ligands [carbonates, phosphates and ethylenediaminetetraacetic acid (EDTA)]. Three fractions (nominal added, dissolved and
free) of cobalt were determined in the liquid media and were correlated with data from batch toxicity experiments. The average
concentration of cobalt that was required for 50% inhibition of methanogenic activity (IC50) for free Co2+ in the three sets of measurements was 13 μmol/L with a standard deviation of 22% and a similarity of 72% between the data
obtained in the three different media for the range of cobalt concentrations investigated. The standard deviation of the IC50 for the other two fractions was much higher, i.e. 85 and 144% for the added cobalt and dissolved cobalt, respectively, and
the similarity was almost 0% for both fractions. Complexation (and precipitation) with EDTA, phosphates and carbonates was
shown to decrease the toxicity of cobalt on methylotrophic methanogenesis. The free cobalt concentration is proposed to be
the key parameter to correlate with cobalt toxicity. Thus, the toxicity of cobalt to granular sludge can be estimated based
on the equilibrium-free cobalt concentration. 相似文献
78.
Omar Domínguez-Domínguez Fernando Alda Gerardo Pérez-Ponce de León José Luis García-Garitagoitia Ignacio Doadrio 《BMC evolutionary biology》2008,8(1):161
Background
Tectonic, volcanic and climatic events that produce changes in hydrographic systems are the main causes of diversification and speciation of freshwater fishes. Elucidate the evolutionary history of freshwater fishes permits to infer theories on the biotic and geological evolution of a region, which can further be applied to understand processes of population divergence, speciation and for conservation purposes. The freshwater ecosystems in Central Mexico are characterized by their genesis dynamism, destruction, and compartmentalization induced by intense geologic activity and climatic changes since the early Miocene. The endangered goodeid Zoogoneticus quitzeoensis is widely distributed across Central México, thus making it a good model for phylogeographic analyses in this area. 相似文献79.
The sequence characterized amplified region (SCAR) marker SCK13(603), associated with ascochyta blight resistance in a chickpea recombinant inbred line (RIL) population, was used as anchored sequence for genome walking. The PCRs performed in the walking steps to walk in the same direction produced eight bands in 5' direction and five bands in 3' direction with a length ranking from 530 to 2,871 bp. The assembly of the bands sequences along with the sequence of SCK13(603) resulted in 7,815 bp contig. Blastn analyses showed stretches of DNA sequence mainly distributed from the nucleotides 1,500 to 4,500 significantly similar to Medicago truncatula genomic DNA. Three open reading frames (ORFs) were identified and blastp analysis of predicted amino acids sequences revealed that ORF1, ORF2 and ORF3 had significant similarity to a CCHC zinc finger protein, to an integrase, and to a precursor of the glucoamylase s1/s2, respectively, from M. truncatula. The high homology of the putative proteins derived from ORF1 and ORF2 with retrotransposon proteins and the prediction of the existence of conserved domains usually present in retrotransposon proteins indicate that the marker SCK13(603) is located in a region of a putative retrotransposon. The information generated in this study has contributed to increase the knowledge of this important region for blight resistance in chickpea. 相似文献
80.
Loza-Mejía MA Maldonado-Hernández K Rodríguez-Hernández F Rodríguez-Sotres R González-Sánchez I Quintero A Solano JD Lira-Rocha A 《Bioorganic & medicinal chemistry》2008,16(3):1142-1149
A series of novel alkylamino and 9-anilinothiazolo[5,4-b]quinolines were synthesized as potential antitumoral agents. The in vitro cytotoxicity of these compounds was evaluated on several cell lines. The inclusion of electron-withdrawn/acceptor hydrogen-bond groups at position 3' of the anilino ring and the presence of an alkylamino chain on the tricyclic framework (regardless of its position) seem to be structural features relevant to cytotoxic activity. 相似文献