136 research outputs found
Ovarian Development of Female-Female Pairs in the Termite, Reticulitermes speratus
In the rhinotermitid termite Reticulitermes speratus (Kolbe) (Isoptera: Rhinotermitidae), facultative parthenogenesis is known to occur occasionally and females cooperate with other females to found the colony. To elucidate the ovarian development in these two females, incipient female-female colonies were established under laboratory conditions, and the process of colony development was observed at 0.5, 1.5, 2.5, 3.5, and 7.5 months (stages I–V, respectively) after colony foundation. Ovarian development, vitellogenin gene expression, and juvenile hormone (JH) titers were examined. A precise reproductive cycle in both females was observed, in which the oviposition rate was relatively higher during stages I and II, decreased during stages III and IV, and then increased again at stage V. JH III titer and vitellogenin gene expression changed in parallel throughout the reproductive cycle of these queens. Ovarian maturation and vitellogenesis were similar in both females in a female-female colony at all stages examined, suggesting that no conflicts existed for two females in terms of oviposition
Light-Induced Nanosecond Relaxation Dynamics of Rhenium-Labeled Pseudomonas aeruginosa Azurins.
Time-resolved phosphorescence spectra of Re(CO)3(dmp)+ and Re(CO)3(phen)+ chromophores (dmp = 4,7-dimethyl-1,10-phenanthroline, phen = 1,10-phenanthroline) bound to surface histidines (H83, H124, and H126) of Pseudomonas aeruginosa azurin mutants exhibit dynamic band maxima shifts to lower wavenumbers following 3-exponential kinetics with 1-5 and 20-100 ns major phases and a 1.1-2.5 μs minor (5-16%) phase. Observation of slow relaxation components was made possible by using an organometallic Re chromophore as a probe whose long phosphorescence lifetime extends the observation window up to ∼3 μs. Integrated emission-band areas also decay with 2- or 3-exponential kinetics; the faster decay phase(s) is relaxation-related, whereas the slowest one [360-680 ns (dmp); 90-140 ns (phen)] arises mainly from population decay. As a result of shifting bands, the emission intensity decay kinetics depend on the detection wavelength. Detailed kinetics analyses and comparisons with band-shift dynamics are needed to disentangle relaxation and population decay kinetics if they occur on comparable timescales. The dynamic phosphorescence Stokes shift in Re-azurins is caused by relaxation motions of the solvent, the protein, and solvated amino acid side chains at the Re binding site in response to chromophore electronic excitation. Comparing relaxation and decay kinetics of Re(dmp)124K122Cu II and Re(dmp)124W122Cu II suggests that electron transfer (ET) and relaxation motions in the W122 mutant are coupled. It follows that nanosecond and faster photo-induced ET steps in azurins (and likely other redox proteins) occur from unrelaxed systems; importantly, these reactions can be driven (or hindered) by structural and solvational dynamics
Hole Hopping Across a Protein-Protein Interface.
We have investigated photoinduced hole hopping in a Pseudomonas aeruginosa azurin mutant Re126WWCuI, where two adjacent tryptophan residues (W124 and W122) are inserted between the CuI center and a Re photosensitizer coordinated to a H126 imidazole (Re = ReI(H126)(CO)3(dmp)+, dmp = 4,7-dimethyl-1,10-phenanthroline). Optical excitation of this mutant in aqueous media (//(CuII)' back ET that occurs over 12 Å, in contrast to the 23 Å, 120 us step in Re126WWCuI. Importantly, dimerization makes Re126FWCuI photoreactive and, in the case of {Re126WWCuI}2, channels the photoproduced "hole" to the molecule that was not initially photoexcited, thereby shortening the lifetime of ReI(H126)(CO)3(dmp•-)//CuII. Whereas two adjacent W124 and W122 indoles dramatically enhance CuI->*Re intramolecular multistep ET, the tryptophan quadruplex in {Re126WWCuI}2 does not accelerate intermolecular electron transport; instead, it acts as a hole storage and crossover unit between inter- and intramolecular ET pathways. Irradiation of {Re126WWCuII}2 or {Re126FWCuII}2 also triggers intermolecular *Re////(W122•+)' intermolecular charge recombination. Our findings shed light on the factors that control interfacial hole/electron hopping in protein complexes and on the role of aromatic amino acids in accelerating long-range electron transport
Two Tryptophans Are Better Than One in Accelerating Electron Flow through a Protein
We
have constructed and structurally characterized a <i>Pseudomonas
aeruginosa</i> azurin mutant <b>Re126WWCu<sup>I</sup></b>, where two adjacent tryptophan residues (W124 and W122, indole separation
3.6–4.1 Å) are inserted between the Cu<sup>I</sup> center
and a Re photosensitizer coordinated to the imidazole of H126 (Re<sup>I</sup>(H126)(CO)<sub>3</sub>(4,7-dimethyl-1,10-phenanthroline)<sup>+</sup>). Cu<sup>I</sup> oxidation by the photoexcited Re label (*Re)
22.9 Å away proceeds with a ∼70 ns time constant, similar
to that of a single-tryptophan mutant (∼40 ns) with a 19.4
Å Re–Cu distance. Time-resolved spectroscopy (luminescence,
visible and IR absorption) revealed two rapid reversible electron
transfer steps, W124 → *Re (400–475 ps, <i>K</i><sub>1</sub> ≅ 3.5–4) and W122 → W124<sup>•+</sup> (7–9 ns, <i>K</i><sub>2</sub> ≅ 0.55–0.75),
followed by a rate-determining (70–90 ns) Cu<sup>I</sup> oxidation
by W122<sup>•+</sup> ca. 11 Å away. The photocycle is
completed by 120 μs recombination. No photochemical Cu<sup>I</sup> oxidation was observed in <b>Re126FWCu<sup>I</sup></b>, whereas
in <b>Re126WFCu<sup>I</sup></b>, the photocycle is restricted
to the ReH126W124 unit and Cu<sup>I</sup> remains isolated. QM/MM/MD
simulations of <b>Re126WWCu<sup>I</sup></b> indicate that indole
solvation changes through the hopping process and W124 → *Re
electron transfer is accompanied by water fluctuations that tighten
W124 solvation. Our finding that multistep tunneling (hopping) confers
a ∼9000-fold advantage over single-step tunneling in the double-tryptophan
protein supports the proposal that hole-hopping through tryptophan/tyrosine
chains protects enzymes from oxidative damage
Correlation Index-Based Responsible-Enzyme Gene Screening (CIRES), a Novel DNA Microarray-Based Method for Enzyme Gene Involved in Glycan Biosynthesis
BACKGROUND: Glycan biosynthesis occurs though a multi-step process that requires a variety of enzymes ranging from glycosyltransferases to those involved in cytosolic sugar metabolism. In many cases, glycan biosynthesis follows a glycan-specific, linear pathway. As glycosyltransferases are generally regulated at the level of transcription, assessing the overall transcriptional profile for glycan biosynthesis genes seems warranted. However, a systematic approach for assessing the correlation between glycan expression and glycan-related gene expression has not been reported previously. METHODOLOGY: To facilitate genetic analysis of glycan biosynthesis, we sought to correlate the expression of genes involved in cell-surface glycan formation with the expression of the glycans, as detected by glycan-recognizing probes. We performed cross-sample comparisons of gene expression profiles using a newly developed, glycan-focused cDNA microarray. Cell-surface glycan expression profiles were obtained using flow cytometry of cells stained with plant lectins. Pearson's correlation coefficients were calculated for these profiles and were used to identify enzyme genes correlated with glycan biosynthesis. CONCLUSIONS: This method, designated correlation index-based responsible-enzyme gene screening (CIRES), successfully identified genes already known to be involved in the biosynthesis of certain glycans. Our evaluation of CIRES indicates that it is useful for identifying genes involved in the biosynthesis of glycan chains that can be probed with lectins using flow cytometry
SUSCEPTIBILITY TO MITOMYCIN C AND LECITHINASE ACTIVITIES OF CLOSTRIDIUM OEDEMATIENS (C. NOVYI) TYPES B AND D
Direct detection of the nitrooxyperoxy radicals in the oxidation of VOCs by NO_3
The photooxidn. of volatile org. compds. (VOCs) is a significant source of secondary org. aerosols (SOA) in the
troposphere. The nighttime chem. leading to SOA growth however remains less characterized. The nitrate radical NO3
is one of the most important nighttime oxidants. In the presence of oxygen, the free radical addn. of NO3 to unsatd.
VOCs will lead to formation of nitrooxyalkyl peroxy radicals. While various studies have measured the further product
yields and SOA formation from the reaction, the peroxy intermediate has yet to be directly obsd. We use cavity ringdown
spectroscopy to directly detect the nitrooxyalkyl peroxy radicals via the A-X electronic absorption. The A-X electronic
transition has enough structure to distinguish between different peroxy radicals as well as different conformers of the
same peroxy radical. In order to elucidate the mechanism and formation of the intermediate, we present results from the
addn. of NO3 to one of the simplest unsatd. VOCs, cis and trans 2-butene
Intra- and intermolecular electron hopping via tryptophan in azurin
We previously showed that electron transfer (ET) between electronically excited Re (^*RE) and Cu(I)in Re-modified Pseudomonas aeruginosa azurin mutant ReH124W122Cu(I)(Re=4,7-dimethylphenanthrolineRe(CO)3) is facilitated by W122, with hopping occurring over 19 Å in 30 ns, >100x faster than single step electron tunneling
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