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1.
《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(12):2331-2337
Conventional empirical methods for the quantification of the helical content of proteins in solution using circular dichroism (CD) primarily rely on spectral data acquired between wavelengths of 190 and 230 nm. The presence of chemical species in a protein solution with strong absorbance within this range can interfere with the ability to use these methods for the determination of the protein's helical structure. The objective of this research was to overcome this problem by developing a method for CD spectral analysis that relies on spectral features above this wavelength range. In this study, we determined that the slopes of CD spectra acquired over the 230 to 240 nm region strongly correlate with the helix contents including α-helix and 310-helix of protein as determined using conventional CD algorithms that rely on wavelengths between 190 and 230 nm. This approach (i.e., the 230–240 nm slope method) is proposed as an effective method to determine the helix content within proteins in the presence of additives such as detergents or denaturants with high absorbance of wavelengths up to 230 nm. 相似文献
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3.
《BBA》2006,1757(9-10):1133-1143
In cytochrome c oxidase, oxido-reductions of heme a/CuA and heme a3/CuB are cooperatively linked to proton transfer at acid/base groups in the enzyme. H+/e− cooperative linkage at Fea3/CuB is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H+/e− linkage at heme a (and CuA). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H2O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/CuA and heme a3/CuB in the soluble oxidase; (iii) H+ release in the external phase (i.e. H+ pumping) associated with the oxidative (R → O transition), reductive (O → R transition) and a full catalytic cycle (R → O → R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H+/e− linkage at heme a/CuA and heme a3/CuB with acid/base clusters, C1 and C2 respectively, and protonmotive steps of the reduction of O2 to water are involved in proton pumping. 相似文献
4.
The complete mitochondrial genome (mitogenome) of Bombyx mori strain Dazao (Lepidoptera: Bombycidae) was determined to be 15,653 bp, including 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a A + T-rich region. It has the typical gene organization and order of mitogenomes from lepidopteran insects. The AT skew of this mitogenome was slightly positive and the nucleotide composition was also biased toward A + T nucleotides (81.31%). All PCGs were initiated by ATN codons, except for cytochrome c oxidase subunit 1 (cox1) gene which was initiated by CGA. The cox1 and cox2 genes had incomplete stop codons consisting of just a T. All the tRNA genes displayed a typical clover-leaf structure of mitochondrial tRNA. The A + T-rich region of the mitogenome was 495 bp in length and consisted of several features common to the lepidopteras. Phylogenetic analysis showed that the B. mori Dazao was close to Bombycidae. 相似文献
5.
《Ecological Engineering》2007,29(3):232-244
Urban wetlands typically have few plant species. In wetlands designed to improve water quality, nutrient-rich water and highly variable water levels often favor aggressive, flood-tolerant plants, such as Typha × glauca (hybrid cattail). At Des Plaines River Wetlands Demonstration Site (Lake Co., IL), we assessed T. × glauca dominance and plant community composition under varying hydroperiods in a complex of eight constructed wetlands. Plots flooded for more than 5 weeks during the growing season tended to be dominated by T. × glauca, while plots flooded fewer days did not. Plots with high cover of T. × glauca had low species richness (negative correlation, R2 = 0.72, p < 0.001). However, overall species richness of the wetland complex was high (94 species), indicating that wetlands in urbanizing landscapes can support many plant species where T. × glauca is not dominant. T. × glauca-dominated areas resisted the establishment of a native plant community. Removing T. × glauca and introducing native species increased diversity initially, but did not prevent re-invasion. Although 12 of the 24 species we seeded became established in our cleared plots, T. × glauca rapidly re-invaded. In year 1, T. × glauca regained an average of 11 ramets m−2, and its density doubled in year 2. The likelihood of planted species surviving decreased as duration of inundation increased, and in both seeded and planted plots, graminoids had greater survivorship through year 2 than forbs across a range of water levels. Within 4 years, however, T. × glauca was the most common plant, present in 92% of the cleared plots. Simply removing T. × glauca and adding propagules to an urban wetland is not sufficient to increase diversity. 相似文献
6.
《BBA》2014,1837(2):315-325
The spectral global quantum yield (YII, electrons/photons absorbed) of photosystem II (PSII) was measured in sunflower leaves in State 1 using monochromatic light. The global quantum yield of PSI (YI) was measured using low-intensity monochromatic light flashes and the associated transmittance change at 810 nm. The 810-nm signal change was calibrated based on the number of electrons generated by PSII during the flash (4 · O2 evolution) which arrived at the PSI donor side after a delay of 2 ms. The intrinsic quantum yield of PSI (yI, electrons per photon absorbed by PSI) was measured at 712 nm, where photon absorption by PSII was small. The results were used to resolve the individual spectra of the excitation partitioning coefficients between PSI (aI) and PSII (aII) in leaves. For comparison, pigment–protein complexes for PSII and PSI were isolated, separated by sucrose density ultracentrifugation, and their optical density was measured. A good correlation was obtained for the spectral excitation partitioning coefficients measured by these different methods. The intrinsic yield of PSI was high (yI = 0.88), but it absorbed only about 1/3 of quanta; consequently, about 2/3 of quanta were absorbed by PSII, but processed with the low intrinsic yield yII = 0.63. In PSII, the quantum yield of charge separation was 0.89 as detected by variable fluorescence Fv/Fm, but 29% of separated charges recombined (Laisk A, Eichelmann H and Oja V, Photosynth. Res. 113, 145–155). At wavelengths less than 580 nm about 30% of excitation is absorbed by pigments poorly connected to either photosystem, most likely carotenoids bound in pigment–protein complexes. 相似文献
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Three tortricid pests, Grapholita dimorpha (Komai), G. molesta (Busck), and Carposina sasakii (Matsumura), are known as internal apple feeders in Korea. To identify young larvae, this study developed two types of molecular markers from their mitochondrial DNA (mtDNA) sequences. To this end, six different loci of mtDNA were sequenced in G. dimorpha: cytochrome oxidase subunit I (460 bp), cytochrome oxidase subunit II (446 bp), cytochrome b (308 bp), NADH dehydrogenase 3 (585 bp), NADH dehydrogenase 4 (ND4, 835 bp), and 16S rRNA (1300 bp). These sequences were compared with those of G. molesta and C. sasakii in order to develop PCR–RFLP and diagnostic primers. ND4 locus was selected to be used for developing a PCR–RFLP marker. ND4-Swa I digests showed two bands for G. dimorpha, one band for G. molesta, and three bands for C. sasakii. On the other hand, species-specific diagnostic PCR primers were developed using ND4 locus. These markers were then applied to diagnose larvae infesting apples to determine species-specific fruit damage patterns, in which G. dimorpha, G. molesta, and C. sasakii showed different feeding behaviors in terms of their main feeding sites in apple fruits. 相似文献
8.
《生物化学与生物物理学报:生物膜》2015,1848(2):488-495
The palmitate/Ca2 +-induced (Pal/Ca2 +) pore, which is formed due to the unique feature of long-chain saturated fatty acids to bind Ca2 + with high affinity, has been shown to play an important role in the physiology of mitochondria. The present study demonstrates that the efflux of Ca2 + from rat liver mitochondria induced by ruthenium red, an inhibitor of the energy-dependent Ca2 + influx, seems to be partly due to the opening of Pal/Ca2 + pores. Exogenous Pal stimulates the efflux. Measurements of pH showed that the Ca2 +-induced alkalization of the mitochondrial matrix increased in the presence of Pal. The influx of Ca2 + (Sr2 +) also induced an outflow of K+ followed by the reuptake of the ion by mitochondria. The outflow was not affected by a K+/H+ exchange blocker, and the reuptake was prevented by an ATP-dependent K+ channel inhibitor. It was also shown that the addition of Sr2 + to mitochondria under hypotonic conditions was accompanied by reversible cyclic changes in the membrane potential, the concentrations of Sr2 + and K+ and the respiratory rate. The cyclic changes were effectively suppressed by the inhibitors of Ca2 +-dependent phospholipase A2, and a new Sr2 + cycle could only be initiated after the previous cycle was finished, indicating a refractory period in the mitochondrial sensitivity to Sr2 +. All of the Ca2 +- and Sr2 +-induced effects were observed in the presence of cyclosporin A. This paper discusses a possible role of Pal/Ca2 + pores in the maintenance of cell ion homeostasis. 相似文献
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《Journal of Asia》2014,17(4):647-651
The veiled polypore, Cryptoporus volvatus, is distributed widely in North America and East Asia and is believed to have a mutualistic relationship with coleopteran species—the fungus providing food and shelter in basidiocarps and beetle helping disperse spores.Seventy fresh basidiocarps of C. volvatus were collected from the Japanese red pine (Pinus densiflora) in the spring season of 2013 from two sites in Korea. A total of 251 insects (101 adult and 150 larvae) were collected from the inside of basidiocarps and identified using morphology and mitochondrial cytochrome c oxidase subunit I (COI) sequences. Six species belonging to five coleopteran families were identified. The number of spores attached to the bodies of adult insects was counted and average spore counts for each of the six species ranged between 1.0 × 104 and 5.2 × 105 spores/individual. Across localities, three species were shared (Aethina suturalis, Trogossita japonica and Parabolitophagus felix) and carried spores at high densities on their bodies, making them more likely to aid in spore dispersal. 相似文献
11.
Jun Sawai Kazushi Matsumoto Taka-aki Saito Yasuhiko Isomura Risei Wada 《International biodeterioration & biodegradation》2009,63(2):196-200
The influence of infrared (IR) radiation on the viability and heat-activation of Bacillus subtilis spores, suspended in phosphate-buffered saline, was investigated. Two types of IR heaters with different spectral distributions were used. Near-infrared (NIR) and far-infrared (FIR) heaters with main wavelengths of approximately 1 μm and 3–6 μm, respectively, were utilized. Although both irradiation treatments decreased the number of B. subtilis colonies at a bulk temperature of approximately 75 °C, the mode of action was clearly different. In the case of the NIR heater, the number of colony-counts decreased gradually. In contrast, use of the FIR heater resulted in heat activation of the spores during the early stage of irradiation at a low bulk temperature (40–60 °C) over several minutes, followed by a decrease in the number of colonies. Consequently, FIR irradiation inactivated 90% of B. subtilis spores more effectively as compared to NIR irradiation for 20 min with a suspension volume of 20 ml and irradiation energy of 7.57 kW m?2. Spore exposure to FIR irradiation accelerated their germination rate in nutrient broth; however, this was not true for treatment with the NIR heater. The absorption IR spectrum of B. subtilis spores indicated that FIR radiation was absorbed easily by the spore cell components and might activate the bioactive substances involved in germination. Even at the same irradiation energy, the influence of infrared radiation on spore germination was dependent on the IR spectral distribution. Bacterial spores undergoing germination lose their resistance to stressors, such as heat, chemicals and ultraviolet rays. FIR heating promotes heat activation and germination, thereby producing vegetative cells that are more susceptible to other killing methods, enabling the killing of bacterial spores at lower stress without product damage. 相似文献
12.
Karima Fadhlaoui-Zid Leyla Knittweis Didier Aurelle Chaala Nafkha Soufia Ezzeddine Fabio Fiorentino Hisham Ghmati Luca Ceriola Othman Jarboui Ferruccio Maltagliati 《Comptes rendus biologies》2012,335(10-11):625-636
The polymorphism of the mitochondrial gene cytochrome oxidase III was studied in the Mediterranean octopus, Octopus vulgaris Cuvier, 1797. A total of 202 specimens from seven sampling sites were analysed with the aim of elucidating patterns of genetic structure in the central Mediterranean Sea and to give an insight into the phylogeny of the Octopus genus. Phylogenetic analyses showed that individuals from the central Mediterranean belong to the O. vulgaris species whose limits should nevertheless be clarified. Concerning genetic structure, two high-frequency haplotypes were present in all locations. The overall genetic divergence (ΦST = 0.05, P < 0.05) indicated a significant genetic structuring in the study area and an AMOVA highlighted a significant break between western and eastern Mediterranean basins (ΦCT = 0.094, P < 0.05). Possible explanations for the observed patterns of genetic structuring are discussed with reference to their relevance for fisheries management. 相似文献
13.
Bhawana Agarwal Amadou K.S. Camara David F. Stowe Zeljko J. Bosnjak Ranjan K. Dash 《BBA》2012,1817(3):453-465
Modulation of mitochondrial free Ca2 + ([Ca2 +]m) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia–reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca2 +]m and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2 mM) on the magnitudes and time-courses of [Ca2 +]m and mitochondrial redox state (NADH), membrane potential (ΔΨm), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10 mM Na+- or K+-pyruvate/malate (NaPM or KPM) or Na+-succinate (NaSuc) followed by additions of isoflurane, 0.5 mM CaCl2 (≈ 200 nM free Ca2 + with 1 mM EGTA buffer), and 250 μM ADP. Isoflurane stepwise: (a) increased [Ca2 +]m in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in ΔΨm and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, ΔΨm, and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, ΔΨm, and matrix contraction with PM substrates. These findings suggest that isoflurane's effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca2 + uptake by inhibiting the Na+/Ca2 + exchanger (NCE), independent of changes in ΔΨm and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca2 +]m, while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level. 相似文献
14.
Lipid oxidation is a widespread phenomenon in foods and other systems of biological origin. Detection methods for early stages of lipid oxidation are in demand to understand the progress of oxidation in space and time. The fluorescence spectrum of the nonpolar fluorescent probe BODIPY665/676 changes upon reacting with peroxyl radicals originating from 2,2′-azobis(2,4-dimethyl)valeronitrile and tert-butoxyl radicals generated from di-tert-butylperoxide. The excitation wavelength of the main peak of BODIPY665/676 was 675 nm in the fluorometer, and 670 nm under the microscope, and the optimum excitation wavelength for the secondary peak of BODIPY665/676 was 580 nm. Advantages of using BODIPY665/676 are fewer problems with autofluorescence and the possibility of combining several fluorescent probes that are excited and emitted at lower wavelengths. However, because of the spectrum of the probe, specific lasers and detectors are needed for optimal imaging under the microscope. Furthermore, BODIPY665/676 is resistant to photobleaching at both excitation wavelengths, 670 and 580 nm. In diffusion studies, BODIPY665/676 is highly lipophilic, remaining in the lipid phase and not diffusing into the aqueous phase or between lipid droplets. 相似文献
15.
《生物化学与生物物理学报:生物膜》2014,1838(5):1338-1343
PbtA, a putative P1B-type ATPase from the Gram-negative soil bacterium Achromobacter xylosoxidans A8 responsible for Pb2 +/Zn2 +/Cd2 +-resistance in Escherichia coli, was heterologously expressed in Saccharomyces cerevisiae. When present in Zn2 +- and Pb2 +/Cd2 +-hypersensitive S. cerevisiae strains CM137 and DTY168, respectively, PbtA was able to restore Zn2 +- and Pb2 +-resistant phenotype. At the same time, the increase of Pb, Zn, and Cd accumulation in yeast was observed. However, Cd2 +-tolerance of the pbtA-bearing yeasts dramatically decreased. The PbtA-eGFP fusion protein was localized primarily in the tonoplast and also in the plasma membrane and the perinuclear region corresponding to the endoplasmic reticulum at later growth stages. This indicates that PbtA protein is successfully incorporated into membranes in yeasts. Since PbtA caused a substantial increase of Pb2 +/Zn2 +-resistance and accumulation in baker's yeast, we propose its further use for the genetic modification of suitable plant species in order to obtain an effective tool for the phytoremediation of sites polluted by toxic transition metals. 相似文献
16.
《Process Biochemistry》2010,45(7):1036-1042
A recombinant strain of Escherichia coli with CYP102A1 gene was developed for the demethylation of colchicine into their derivatives. The CYP102A1 gene responsible for demethylation was isolated from Bacillus megaterium ACBT03 and amplified using suitable primers. The amplified product was cloned into pET28a+ expression vector using host E. coli BL21(DE3) cells. The CYP3A4 (product of CYP102A1 gene) protein expression and other parameters like substrate toxicity, product toxicity and enzyme activity were optimized in shake flasks; and further scaled-up to 5 l bioreactor with 3 l working volume. In 5 l bioreactor, dissolved oxygen (DO) was optimized for maximum specific growth and enhanced 3-demethylated colchicine (3-DMC) production. The optimized conditions from shake flasks were scaled-up to 70 l bioreactor and resulted into ∼80% conversion of 20 mM colchicine in 48 h with a volumetric productivity of 6.62 mg l−1 h−1. Scale-up factors were measured as volumetric oxygen transfer coefficient (kLa) i.e., 56 h−1 and impeller tip velocity (Vtip) i.e., 7.065 m s−1, respectively. The kinetic parameters Km, kcat, and kcat/Km of the CYP3A4 enzyme using colchicine as the substrate were determined to be 271 ± 30 μM, 8533 ± 25 min−1, and 31.49 μM min−1, respectively, when IPTG induced recombinant E. coli culture was used. 相似文献
17.
E. Sierra-Campos I. Velázquez D. Matuz-Mares A. Villavicencio-Queijeiro J.P. Pardo 《Mitochondrion》2009,9(2):96-102
The mitochondrial respiratory chain of Ustilago maydis contains two terminal oxidases, the cytochrome c oxidase (COX) and the alternative oxidase (AOX). To understand the biochemical events that control AOX activity, we studied the regulation and function of AOX under oxidative stress. The activity of this enzyme was increased by both pyruvate (K05 = 2.6 mM) and purine nucleotides (AMP, K05 = 600 μM) in mitochondria using succinate as respiratory substrate. When U. maydis cells were grown in the presence of antimycin A, the amount of AOX in mitochondria was markedly increased and its selectivity towards AMP and pyruvate changed, suggesting that post-translational events may play a role in the regulation of AOX activity under stress conditions. Addition of antimycin A to isolated mitochondria induced the inactivation of AOX, the formation of lipid peroxides and the loss of glutathione from mitochondria. The two last processes are probably related with the time dependent inactivation of AOX, in agreement with the inhibition of the enzyme by tert-butyl hydroperoxide. Our results suggest that the in vivo operation of AOX in U. maydis depends on the mitochondrial antioxidant machinery, including the glutathione linked systems. 相似文献
18.
《BBA》2006,1757(9-10):1122-1132
The PM → F transition of the catalytic cycle of cytochrome c oxidase from bovine heart was investigated using single-electron photoreduction and monitoring the subsequent events using spectroscopic and electometric techniques. The PM state of the oxidase was generated by exposing the oxidized enzyme to CO plus O2. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a3 with a time constant of about 0.3 ms, as indicated by transients at 605 nm and 580 nm. This rate is ∼ 5-fold more rapid than the rate of electron transfer from heme a to heme a3 in the F → O transition, but is significantly slower than formation of the F state from the PR intermediate in the reaction of the fully reduced enzyme with O2 to form state F (70–90 μs). The ∼ 0.3 ms PM → F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the PM → F transition is generated much later, with a time constant of 1.3 ms. In addition, the PM → F transition of the R. sphaeroides oxidase was also measured and also was shown to have two phases of electrogenic proton transfer, with τ values of 0.18 and 0.85 ms. 相似文献
19.
Melissa A. Liriano Kristen M. Varney Nathan T. Wright Cassandra L. Hoffman Eric A. Toth Rieko Ishima David J. Weber 《Journal of molecular biology》2012,423(3):365-385
Mutations in the second EF-hand (D61N, D63N, D65N, and E72A) of S100B were used to study its Ca2 + binding and dynamic properties in the absence and presence of a bound target, TRTK-12. With D63NS100B as an exception (D63NKD = 50 ± 9 μM), Ca2 + binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (D61NKD = 412 ± 67 μM, D65NKD = 968 ± 171 μM, and E72AKD = 471 ± 133 μM), when compared to wild-type protein (WTKD = 56 ± 9 μM). For the TRTK-12 complexes, the Ca2 +-binding affinity to wild type (WT + TRTKKD = 12 ± 10 μM) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target (D61N + TRTKKD = 29 ± 1.2 μM, D63N + TRTKKD = 10 ± 2.2 μM, D65N + TRTKKD = 73 ± 4.4 μM, and E72A + TRTKKD = 18 ± 3.7 μM). In addition, Rex, as measured using relaxation dispersion for side‐chain 15N resonances of Asn63 (D63NS100B), was reduced upon TRTK-12 binding when measured by NMR. Likewise, backbone motions on multiple timescales (picoseconds to milliseconds) throughout wild type, D61NS100B, D63NS100B, and D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca2 +-bound (2.0 Å) and TRTK-bound (1.2 Å) D63NS100B showed no change in Ca2 + coordination; thus, these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca2 +-binding affinity in solution. Therefore, a model for how S100B–TRTK‐12 complex formation increases Ca2 + binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12. 相似文献
20.
Loss-of-function mutations in PINK1 or parkin genes are associated with juvenile-onset autosomal recessive forms of Parkinson disease. Numerous studies have established that PINK1 and parkin participate in a common mitochondrial-quality control pathway, promoting the selective degradation of dysfunctional mitochondria by mitophagy. Upregulation of parkin mRNA and protein levels has been proposed as protective mechanism against mitochondrial and endoplasmic reticulum (ER) stress. To better understand how parkin could exert protective function we considered the possibility that it could modulate the ER–mitochondria inter-organelles cross talk. To verify this hypothesis we investigated the effects of parkin overexpression on ER–mitochondria crosstalk with respect to the regulation of two key cellular parameters: Ca2 + homeostasis and ATP production. Our results indicate that parkin overexpression in model cells physically and functionally enhanced ER–mitochondria coupling, favored Ca2 + transfer from the ER to the mitochondria following cells stimulation with an 1,4,5 inositol trisphosphate (InsP3) generating agonist and increased the agonist-induced ATP production. The overexpression of a parkin mutant lacking the first 79 residues (ΔUbl) failed to enhance the mitochondrial Ca2 + transients, thus highlighting the importance of the N-terminal ubiquitin like domain for the observed phenotype. siRNA-mediated parkin silencing caused mitochondrial fragmentation, impaired mitochondrial Ca2 + handling and reduced the ER–mitochondria tethering. These data support a novel role for parkin in the regulation of mitochondrial homeostasis, Ca2 + signaling and energy metabolism under physiological conditions. 相似文献