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981.
Hayama K Ishibashi H Ishijima SA Niimi K Tansho S Ono Y Monk BC Holmes AR Harding DR Cannon RD Abe S 《FEMS microbiology letters》2012,328(2):130-137
Clinical management of patients undergoing treatment of oropharyngeal candidiasis with azole antifungals can be impaired by azole resistance. High-level azole resistance is often caused by the overexpression of Candida albicans efflux pump Cdr1p. Inhibition of this pump therefore represents a target for combination therapies that reverse azole resistance. We assessed the therapeutic potential of the D-octapeptide derivative RC21v3, a Cdr1p inhibitor, in the treatment of murine oral candidiasis caused by either the azole-resistant C. albicans clinical isolate MML611 or its azole-susceptible parental strain MML610. RC21v3, fluconazole (FLC), or a combination of both drugs were administered orally to immunosuppressed ICR mice at 3, 24, and 27 h after oral inoculation with C. albicans. FLC protected the mice inoculated with MML610 from oral candidiasis, but was only partially effective in MML611-infected mice. The co-application of RC21v3 (0.02 μmol per dose) potentiated the therapeutic performance of FLC for mice infected with either strain. It caused a statistically significant decrease in C. albicans cfu isolated from the oral cavity of the infected mice and reduced oral lesions. RC21v3 also enhanced the therapeutic activity of itraconazole against MML611 infection. These results indicate that RC21v3 in combination with azoles has potential as a therapy against azole-resistant oral candidiasis. 相似文献
982.
Ishibashi M Uchino M Arai S Kuroki R Arakawa T Tokunaga M 《Archives of biochemistry and biophysics》2012,525(1):47-52
Nucleoside diphosphate kinase (HsNDK) from extremely halophilic haloarchaeon, Halobacterium salinarum, requires salt at high concentrations for folding. A D148C mutant, in which Asp148 was replaced with Cys, was designed to enhance stability and folding in low salt solution by S-S bond. It showed increased thermal stability by about 10 °C in 0.2 M NaCl over the wild type HsNDK. It refolded from heat-denaturation even in 0.1 M NaCl, while the wild type required 2 M NaCl to achieve the same level of activity recovery. This enhanced refolding is due to the three S-S bonds between two basic dimeric units in the hexameric HsNDK structure, indicating that assembly of the dimeric unit may be the rate-limiting step in low salt solution. Circular dichroism and native-PAGE analysis showed that heat-denatured HsNDK formed partially folded dimeric structure, upon refolding, in the absence of salt and the native-like secondary structure in the presence of salt above 0.1 M NaCl. However, it remained dimeric upon prolonged incubation at this salt concentration. In contrary, heat-denatured D148C mutant refolded into tetrameric folding intermediate in the absence of salt and native-like structure above 0.1 M salt. This native-like structure was then converted to the native hexamer with time. 相似文献
983.
Recent neutron diffraction studies on photoactive yellow protein (PYP) proposed that the H bond between protonated Glu46 and the chromophore-ionized p-coumaric acid (pCA) is a low-barrier H bond (LBHB) mainly because the H atom position was assigned at the midpoint of the O(Glu46)-O(pCA) bond. However, the (1)H nuclear magnetic resonance (NMR) chemical shift (δ(H)) was 15.2 ppm, which is lower than the values of 17-19 ppm for typical LBHBs. We evaluated the dependence of δ(H) on an H atom position in the O(Glu46)-O(pCA) bond in the PYP ground state by using a quantum mechanical/molecular mechanical (QM/MM) approach. The calculated chemical shift unambiguously suggested that a δ(H) of 15.2 ppm for the O(Glu46)-O(pCA) bond in NMR studies should correspond to the QM/MM geometry (δ(H) = 14.5 ppm), where the H atom belongs to the Glu moiety, rather than the neutron diffraction geometry (δ(H) = 19.7 ppm), where the H atom is near the midpoint of the donor and acceptor atoms. 相似文献
984.
Nishiyama T Kuroda S Takiguchi E Nakamura T Hashimoto K Tsuzuranuki K Kawakami T Masuho Y Kohroki J 《Biochemical and biophysical research communications》2012,420(3):487-491
ASB2 proteins are E3 ubiquitin (Ub) ligases that ubiquitinate filamins. There are two ASB2 splice variants, ASB2α and ASB2β. ASB2β has a ubiquitin-binding motif (UIM) at the N-terminal region but ASB2α does not. Here, we provide the first evidence that ASB2β but not ASB2α is monoubiquitinated and that this monoubiquitination involves the UIM. Myc-tagged ASB2β and hemagglutinin (HA)-tagged Ub were co-expressed in HEK293 cells using the pCMV expression vector. Immunoprecipitation with an anti-Myc antibody followed by immunoblotting with anti-Myc and anti-HA antibodies showed an additional ASB2β protein band that had both a Myc and a HA tag. The molecular weight of this protein was larger than that of ASB2β, and the difference in molecular weight between these two proteins corresponded to the molecular weight of monoubiquitin, strongly implying that monoubiquitinated ASB2β is produced in cells. ASB2β with mutations in the UIM motif; either Glu·Asp·Glu27-29Ala·Ala·Ala mutations (ASB2β M1) or a Ser38Ala mutation, (ASB2β M2) were not monoubiquitinated, suggesting the importance of the UIM for ASB2β monoubiquitination. Furthermore, an ASB2β mutant that lacked a SOCS box (ASB2β ΔC) and did not show E3 Ub ligase activity was monoubiquitinated to the same extent as the wild-type ASB2β. In contrast, an ASB2β mutant that lacked the UIM-containing domain (ASB2β ΔN) was not monoubiquitinated. These results suggest that ASB2β but not ASB2α might be monoubiquitinated and that the ASB2β UIM motif, but not its E3 Ub ligase activity, plays a pivotal role in this monoubiquitination. 相似文献
985.
986.
Xiang H Ishibashi K Nishikiori M Jaudal MC Ishikawa M Katoh E 《Protein expression and purification》2012,81(1):89-95
Tomato mosaic virus (genus, Tobamovirus) is a member of the alphavirus-like superfamily of positive-strand RNA viruses, which include many plant and animal viruses of agronomical and clinical importance. The RNA of alphavirus-like superfamily members encodes replication-associated proteins that contain a putative superfamily 1 helicase domain. To date, a viral three-dimensional superfamily 1 helicase structure has not been solved. For the study reported herein, we expressed tomato mosaic virus replication proteins that contain the putative helicase domain and additional upstream N-terminal residues in Escherichia coli. We found that an additional 155 residues upstream of the N-terminus of the helicase domain were necessary for stability. We developed an efficient procedure for the expression and purification of this fragment and have examined factors that affect its stability. Finally, we also showed that the stable fragment has nucleoside 5'-triphosphatase activity. 相似文献
987.
Noriko Hiroi Michael Klann Keisuke Iba Pablo de Heras Ciechomski Shuji Yamashita Akito Tabira Takahiro Okuhara Takeshi Kubojima Yasunori Okada Kotaro Oka Robin Mange Michael Unger Akira Funahashi Heinz Koeppl 《EURASIP Journal on Bioinformatics and Systems Biology》2012,2012(1):7
In our previous study, we introduced a combination methodology of
Fluorescence Correlation Spectroscopy (FCS) and Transmission Electron
Microscopy (TEM), which is powerful to investigate the effect of
intracellular environment to biochemical reaction processes. Now, we
developed a reconstruction method of realistic simulation spaces based on
our TEM images. Interactive raytracing visualization of this space allows
the perception of the overall 3D structure, which is not directly accessible
from 2D TEM images. Simulation results show that the diffusion in such
generated structures strongly depends on image post-processing. Frayed
structures corresponding to noisy images hinder the diffusion much stronger
than smooth surfaces from denoised images. This means that the correct
identification of noise or structure is significant to reconstruct
appropriate reaction environment in silico in order to estimate
realistic behaviors of reactants in vivo. Static structures lead to
anomalous diffusion due to the partial confinement. In contrast, mobile
crowding agents do not lead to anomalous diffusion at moderate crowding
levels. By varying the mobility of these non-reactive obstacles (NRO), we
estimated the relationship between NRO diffusion coefficient
(Dnro) and the anomaly in the tracer diffusion
(α). For Dnro=21.96 to 44.49
μm2/s, the simulation results
match the anomaly obtained from FCS measurements. This range of the
diffusion coefficient from simulations is compatible with the range of the
diffusion coefficient of structural proteins in the cytoplasm. In addition,
we investigated the relationship between the radius of NRO and anomalous
diffusion coefficient of tracers by the comparison between different
simulations. The radius of NRO has to be 58 nm when the polymer moves with
the same diffusion speed as a reactant, which is close to the radius of
functional protein complexes in a cell. 相似文献
988.
Periplasmic metal binding protein characterized by high histidine content was cloned from moderate halophile, Chromohalobacter salexigens. The protein, termed histidine-rich metal binding protein (HP), was expressed in and purified from E. coli as a native form. HP bound to Ni- and Cu-loaded chelate columns with high affinity, and Co- and Zn-columns with moderate
affinity. Although the secondary structure was not grossly altered by the addition of 0.2–2.0 M NaCl, the thermal transition
pattern was considerably shifted to higher temperature with increasing salt concentration: melting temperature was raised
by ~20 °C at 2.0 M NaCl over the melting temperature at 0.2 M NaCl. HP showed reversible refolding from thermal melting in
0.2–1.15 M NaCl, while it formed irreversible aggregates upon thermal melting at 2 M NaCl. Addition of 0.01–0.1 mM NiSO4 stabilized HP against thermal melting with high reversibility, while addition above 0.5 mM resulted in irreversible melting
due to aggregation. 相似文献
989.
Masakado Kawata Hiroshi Sawada Jun Yokoyama Lázaro M. Echenique-Díaz Yasuyuki Ishibashi 《Hydrobiologia》2005,533(1-3):229-241
We examined the growth and reproductive rates of freshwater snails, Physa acuta, in two habitat types. In the Asabata habitat, snails lived in isolated water pools, which occasionally joined to form a single large pool; in the Kakegawa habitat, they lived in a slow-running water way. Genetic structure assessments using three microsatellite loci supports the idea that a stable panmictic population occupies the Kakegawa habitat. The Asabata habitat, however, is occupied with an alternate mixing population as revealed by microsatellite data. The Asabata population might alternate between localized mating within isolated pools (as revealed by high F
IS and F
IT values) when the water levels are low and panmixia (as revealed by the low F
ST values and AMOVA analysis) when the habitat is flooded. Laboratory experiments, using snails collected from the two habitats, showed that juvenile snails grew faster, laid more eggs, and laid them earlier in the Asabata habitat than in the Kakegawa habitat. Growth rates were lower at high density than at low density in the Kakegawa habitat; the inverse was true in the Asabata habitat. Density-dependent response of individual snail reproduction was higher in the Kakagawa habitat than in the Asabata habitats. The results support the hypothesis that spatial structure affects the evolution of density-dependent growth rates and of timing for reproduction. 相似文献
990.