The significance of the flexible loop in the azurin (Az-iso2) from the obligate methylotroph Methylomonas sp. strain J

The obligate methylotroph Methylomonas sp. strain J produces two azurins (Az-iso1 and Az-iso2) as candidates for electron acceptor from methylamine dehydrogenase (MADH) in the electron-transfer process involving the oxidation of methylamine to formaldehyde and ammonia. The X-ray crystallographic study indicated that Az-iso2 gives two types of crystals (form I and form II) with polyethylene glycol (PEG4000) and ammonium sulfate as the precipitants, respectively. Comparison between the two Az-iso2 structures in forms I and II reveals the remarkable structural changes at the top surface of the molecule around the copper atom. Az-iso2 possesses Gly43 instead of Val43 or Ala43, which is unique among all other azurins around the copper ligand His46, inducing the remarkable structural change in the loop region from Gly37 to Gly43. When the structure of Az-iso2 is superimposed on that of amicyanin in the ternary complex composed of MADH, amicyanin, and cytochrome c(551), the loop of Az-iso2 deeply overlaps with the light subunit of MADH. However, the Az-iso2 molecule is probably able to avoid any steric hindrance with the cognate MADH to form the complex for intermolecular electron-transfer reaction, since the loop containing Gly43 is flexible. We discuss why the electron-transfer activity of Az-iso2 is fivefold higher than that of Az-iso1.     

Regulation of food provisioning and parental body condition in Leach’s storm-petrels, Oceanodroma leucorhoa: Experimental manipulation of offspring food demand

We examined the effects of offspring food demand on parental regulation of food provisioning and body condition in a small long-lived seabird, Leachs storm-petrels (Oceanodroma leucorhoa). In one experimental group, food demand of chicks on their parents was increased by removing one parent (single), and in another group these food demands were decreased by supplementary feeding of chicks (supplement). A further unmanipulated group provided a control. Feeding frequencies by one parent were higher in the single but lower in the supplement than in the control group, in accordance with the food demand of chicks. The size of meals appeared to be not different among the experimental groups. However, as single parents did not compensate perfectly for the increase of chick food demand by food provisioning, single chicks grew at slower rates and fledged at smaller masses than control chicks. Supplement chicks grew at similar rates and fledged at similar masses as control chicks, because parents decreased food provisioning and food processing capacity of the chicks might be limited. The body condition of parents, which was determined by body mass loss and feather regrowth rate, did not differ among the groups. These results indicate that feeding frequency was regulated by parental decision in this storm-petrel species. Parents may adjust their food provisioning to match the food demand of chicks but within a certain range so as not to deteriorate their own body condition.     

Functional characterization of a new p53 mutant generated by homozygous deletion in a neuroblastoma cell line

p53 is a key modulator of a variety of cellular stresses. In human neuroblastomas, p53 is rarely mutated and aberrantly expressed in cytoplasm. In this study, we have identified a novel p53 mutant lacking its COOH-terminal region in neuroblastoma SK-N-AS cells. p53 accumulated in response to cisplatin (CDDP) and thereby promoting apoptosis in neuroblastoma SH-SY5Y cells bearing wild-type p53, whereas SK-N-AS cells did not undergo apoptosis. We found another p53 (p53DeltaC) lacking a part of oligomerization domain and nuclear localization signals in SK-N-AS cells. p53DeltaC was expressed largely in cytoplasm and lost the transactivation function. Furthermore, a 3'-part of the p53 locus was homozygously deleted in SK-N-AS cells. Thus, our present findings suggest that p53 plays an important role in the DNA-damage response in certain neuroblastoma cells and it seems to be important to search for p53 mutations outside DNA-binding domain.     

Characterization and function of Met150Gln mutant of copper-containing nitrite reductase from Achromobacter cycloclastes IAM1013

The mutant (M150Q-NIR) replacing the Met150 ligand of the type 1 Cu center in Achromobacter cycloclastes nitrite reductase (AcNIR) with Gln has been physicochemically and functionally characterized. The electronic absorption and CD spectra of M150Q-NIR are similar to those of mavicyanin and stellacyanin having the 2His, Cys, and Gln ligands, but the EPR signal has an axial character, although their blue copper proteins show rhombic EPR signals. The mutant has about 80% catalytic activity of AcNIR. Moreover, the midpoint potential (E(1/2)) of M150Q-NIR is +113 mV vs. NHE at pH 7.0, being negatively shifted compared to that of AcNIR (+240 mV). Although the intermolecular electron-transfer process from Achromobacter cycloclastes pseudoazurin (pAz) to M150Q-NIR was not detected, the pAz mutant (M86Q-pAz) replacing the Met86 ligand with Gln transfers one electron to the NIR mutant with an intermolecular electron-transfer rate constant (k(ET)) of 2.3 x 10(5)M(-1)s(-1).     

Movements and activities of male black‐tailed gulls in breeding and sabbatical years

Long‐lived animals sometimes skip one or more breeding seasons; however, little is known about their movements and activities during such ‘sabbatical’ periods. Here we present novel data on year‐round movements and activities of two male black‐tailed gulls Larus crassirostris during a sabbatical year. We compare the data with those in a year when they bred and with those of two other breeding males. The year‐round migration routes of two sabbatical males were consistent with those of the breeding males: they returned to the breeding area but did not visit the colony in the sabbatical year. They landed more frequently on water (a potential index of foraging effort) during the non‐breeding autumn and winter prior to the sabbatical year than before breeding. Sabbatical gulls may forage more intensively to recover body condition immediately after breeding.     

Epigenetic response of endothelial cells to different wall shear stress magnitudes: A report of new mechano-miRNAs

Endothelial cells (ECs) respond to flow stress via a variety of mechanisms, leading to various intracellular responses that can modulate the vessel wall and lead to diseases if the flow is disturbed. Mechano-microRNAs (miRNAs) are a subset of miRNAs in the ECs that are flow responsive. Mechano-miRNAs were shown to be related to atherosclerosis pathophysiology, and a number of them were identified as pathologic. Here, we exposed human carotid ECs to different wall shear stresses (WSS), high and low, and evaluated the response of miRNAs by microarray and quantitative polymerase chain reaction analysis. We discovered five new mechano-miRNAs that were not reported in that context previously to the best of our knowledge. Moreover, functional pathway analysis revealed that under low WSS conditions, several pathways regulating apoptosis are affected. In addition, KLF2 and KLF4, known atheroprotective genes, were downregulated under low WSS and upregulated under high WSS. KLF2 and VCAM1, both angiogenic, were upregulated under high WSS. NOS3, which is vascular protective, was also upregulated with higher WSS. On the contrary, ICAM-1 and E-selectin, both atherogenic and proinflammatory, were upregulated with high WSS. Collectively, the epigenetic landscape with the gene expression analysis reveals that low WSS is associated with a proapoptotic state, while high WSS is associated with a proliferative and proinflammatory state.     

Measurement of Technetium-99m Sestamibi Signals in Rats Administered a Mitochondrial Uncoupler and in a Rat Model of Heart Failure

Background

Many methods have been used to assess mitochondrial function. Technetium-99m sestamibi (^99mTc-MIBI), a lipophilic cation, is rapidly incorporated into myocardial cells by diffusion and mainly localizes to the mitochondria. The purpose of this study was to investigate whether measurement of ^99mTc-MIBI signals in animal models could be used as a tool to quantify mitochondrial membrane potential at the organ level.

Methods and Results

We analyzed ^99mTc-MIBI signals in Sprague-Dawley (SD) rat hearts perfused with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler known to reduce the mitochondrial membrane potential. ^99mTc-MIBI signals could be used to detect changes in the mitochondrial membrane potential with sensitivity comparable to that obtained by two-photon laser microscopy with the cationic probe tetramethylrhodamine ethyl ester (TMRE). We also measured ^99mTc-MIBI signals in the hearts of SD rats administered CCCP (4 mg/kg intraperitoneally) or vehicle. ^99mTc-MIBI signals decreased in rat hearts administered CCCP, and the ATP content, as measured by ³¹P magnetic resonance spectroscopy, decreased simultaneously. Next, we administered ^99mTc-MIBI to Dahl salt-sensitive rats fed a high-salt diet, which leads to hypertension and heart failure. The ^99mTc-MIBI signal per heart tissue weight was inversely correlated with heart weight, cardiac function, and the expression of atrial natriuretic factor, a marker of heart failure, and positively correlated with the accumulation of labeled fatty acid analog. The ^99mTc-MIBI signal per liver tissue weight was lower than that per heart tissue weight.

Conclusion

Measurement of ^99mTc-MIBI signals can be an effective tool for semiquantitative investigation of cardiac mitochondrial membrane potential in the SD rat model by using a chemical to decrease the mitochondrial membrane potential. The ^99mTc-MIBI signal per heart tissue weight was inversely correlated with the severity of heart failure in the Dahl rat model.     

Structure-based engineering of Alcaligenes xylosoxidans copper-containing nitrite reductase enhances intermolecular electron transfer reaction with pseudoazurin

The intermolecular electron transfer from Achromobacter cycloclastes pseudoazurin (AcPAZ) to wild-type and mutant Alcaligenes xylosoxidans nitrite reductases (AxNIRs) was investigated using steady-state kinetics and electrochemical methods. The affinity and the electron transfer reaction constant (k(ET)) are considerably lower between AcPAZ and AxNIR (K(m) = 1.34 mM and k(ET) = 0.87 x 10(5) M(-1) s(-1)) than between AcPAZ and its cognate nitrite reductase (AcNIR) (K(m) = 20 microM and k(ET) = 7.3 x 10(5) M(-1) s(-1)). A negatively charged hydrophobic patch, comprising seven acidic residues around the type 1 copper site in AcNIR, is the site of protein-protein interaction with a positively charged hydrophobic patch on AcPAZ. In AxNIR, four of the negatively charged residues (Glu-112, Glu-133, Glu-195, and Asp-199) are conserved at the corresponding positions of AcNIR, whereas the other three residues are not acidic amino acids but neutral amino acids (Ala-83, Ala-191, and Gly-198). Seven mutant AxNIRs with additional negatively charged residues surrounding the hydrophobic patch of AxNIR (A83D, A191E, G198E, A83D/A191E, A93D/G198E, A191E/G198E, and A83D/A191E/G198E) were prepared to enhance the specificity of the electron transport reaction between AcPAZ and AxNIR. The k(ET) values of these mutants become progressively larger as the number of mutated residues increases. The K(m) and k(ET) values of A83D/A191E/G198E (K(m) = 88 microM and k(ET) = 4.1 x 10(5) M(-1) s(-1)) are 15-fold smaller and 4.7-fold larger than those of wild-type AxNIR, respectively. These results suggest that the introduction of negatively charged residues into the docking surface of AxNIR facilitates both the formation of electron transport complex and the electron transfer reaction.     

Co(II)-substituted Octopus vulgaris hemocyanin

Co(II)-substituted hemocyanin (Co(II)Hc) of the octopus, Octopus vulgaris, has been prepared by dialysis of apohemocyanin against Co(II·) ion and subsequent Chelex-treatment. The blue 50%-Co(II)Hc (half-apo Co(II)Hc), in which binuclear coppers are replaced in the hemocyanin by a single Co(II), exhibits two absorption maxima at 560 (?_Co=250) and 594 nm (?_Co=320 M^?1 cm^?1) and a shoulder near 610 nm, all of which are attributed to a dd transition of high spin Co(II) (S=3/2) with a tetrahedral geometry. The magnetic circular dichroism (MCD) spectrum in this region also suggests the existence of a tetrahedral Co(II) species in the protein. The visible absorption and MCD spectra of octopus 50%-Co(II)Hc are quite similar to those of squid 50%-Co(II)Hc described in the previous paper (S. Suzuki, J. Kino, M. Kimura, W. Mori and A. Nakahara, Inorg. Chim. Acta, 66, 41 (1982)). The formation of half-apo Co(II)Hc demonstrates that the binuclear copper sites in native octopus hemocyanin may differ from each other in coordination geometry, as in other molluscan hemocyanins, squid and snail hemocyanins. The coordination environment of the active-site Co(II) substituted for Cu in the octopus hemocyanin is the same as that of the corresponding active site of the squid hemocyanin.     

Characterization of two Cu-containing protein fragments obtained by limited proteolysis of Hyphomicrobiumdenitrificans A3151 nitrite reductase

The unusual Hyphomicrobium denitrificans nitrite reductase containing two type 1 Cu sites and one type 2 Cu site (MW, 50 kDa) has been proteolyzed to two protein fragments (14 and 35 kDa) with subtilisin. The visible absorption, CD, and EPR spectra of these proteins imply that the blue 14-kDa protein fragment has one type 1 Cu site, which is axially elongated trigonal bipyramidal, and the green 35-kDa protein fragment has one type 1 Cu site having a flattened tetrahedral geometry with one type 2 Cu site. The 35-kDa fragment shows the nitrite reduction activity a little higher than to that of native HdNIR. The redox potentials of the 14- and 35-kDa fragments are +345 and +353mV vs. NHE at pH 7.0, respectively. Moreover, the intermolecular electron transfer rate constant of the 35-kDa fragment from an electron donor, cognate cytochrome c(550), is nearly the same as that of the native enzyme.