Effect of a combination of astaxanthin supplementation, heat stress, and intermittent reloading on satellite cells during disuse muscle atrophy.

We examined the effect of a combination of astaxanthin (AX) supplementation, repeated heat stress, and intermittent reloading (IR) on satellite cells in unloaded rat soleus muscles. Forty-nine male Wistar rats (8-week-old) were divided into control, hind-limb unweighting (HU), IR during HU, IR with AX supplementation, IR with repeated heat stress (41.0-41.5 °C for 30 min), and IR with AX supplementation and repeated heat stress groups. After the experimental period, the antigravitational soleus muscle was analyzed using an immunohistochemical technique. Our results revealed that the combination of dietary AX supplementation and heat stress resulted in protection against disuse muscle atrophy in the soleus muscle. This protective effect may be partially due to a higher satellite cell number in the atrophied soleus muscle in the IR/AX/heat stress group compared with the numbers found in the other groups. We concluded that the combination treatment with dietary AX supplementation and repeated heat stress attenuates soleus muscle atrophy, in part by increasing the number of satellite cells.

g-Tensor Directions in the Protein Structural Frame of Hyperthermophilic Archaeal Reduced Rieske-Type Ferredoxin Explored by 13C Pulsed Electron Paramagnetic Resonance.

Interpretation of magnetic resonance data in the context of structural and chemical biology requires prior knowledge of the g-tensor directions for paramagnetic metallo-cofactors with respect to the protein structural frame. Access to this information is often limited by the strict requirement of suitable protein crystals for single-crystal electron paramagnetic resonance (EPR) measurements or the reliance on protons (with ambiguous locations in crystal structures) near the paramagnetic metal site. Here we develop a novel pulsed EPR approach with selective 13Cß-cysteine labeling of model [2Fe-2S] proteins to help bypass these problems. Analysis of the 13Cß-cysteine hyperfine tensors reproduces the g-tensor of the Pseudomonas putida ISC-like [2Fe-2S] ferredoxin (FdxB). Its application to the hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin (ARF) from Sulfolobus solfataricus, for which the single-crystal EPR approach was not feasible, supports the best-fit g x-, g z-, and g y-tensor directions of the reduced cluster as nearly along Fe-Fe, S-S, and the cluster plane normal, respectively. These approximate principal directions of the reduced ARF g-tensor, explored by 13C pulsed EPR, are less skewed from the cluster molecular axes and are largely consistent with those previously determined by single-crystal EPR for the cytochrome bc1-associated, reduced Rieske [2Fe-2S] center. This suggests the approximate g-tensor directions are conserved across the phylogenetically and functionally divergent Rieske-type [2Fe-2S] proteins.

Dietary astaxanthin supplementation attenuates disuse-induced muscle atrophy and myonuclear apoptosis in the rat soleus muscle.

Extended periods of skeletal muscle disuse results in muscle atrophy and weakness. Currently, no therapeutic treatment is available for the prevention of this problem. Nonetheless, growing evidence suggests that prevention of disuse-induced oxidative stress in inactive muscle fibers can delay inactivity-induced muscle wasting. Therefore, this study tested the hypothesis that dietary supplementation with the antioxidant astaxanthin would protect against disuse muscle atrophy, in part, by prevention of myonuclear apoptosis. Wistar rats (8 weeks old) were divided into control (CT, n = 9), hindlimb unloading (HU, n = 9), and hindlimb unloading with astaxanthin (HU + AX, n = 9) groups. Following 2 weeks of dietary supplementation, rats in the HU and HU + AX groups were exposed to unloading for 7 days. Seven-day unloading resulted in reduced soleus muscle weight and myofiber cross-sectional area (CSA) by ~30 and ~47 %, respectively. Nonetheless, relative muscle weights and CSA of the soleus muscle in the HU + AX group were significantly greater than those of the HU group. Moreover, astaxanthin prevented disuse-induced increase in the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei. We conclude that astaxanthin supplementation prior to and during hindlimb unloading attenuates soleus muscle atrophy, in part, by suppressing myonuclear apoptosis.

Astaxanthin intake attenuates muscle atrophy caused by immobilization in rats.

Astaxanthin is a carotenoid pigment and has been shown to be an effective inhibitor of oxidative damage. We tested the hypothesis that astaxanthin intake would attenuate immobilization-induced muscle atrophy in rats. Male Wistar rats (14-week old) were fed for 24 days with either astaxanthin or placebo diet. After 14 days of each experimental diet intake, the hindlimb muscles of one leg were immobilized in plantar flexion position using a plaster cast. Following 10 days of immobilization, both the atrophic and the contralateral plantaris muscles were removed and analyzed to determine the level of muscle atrophy along with measurement of the protein levels of CuZn-superoxide dismutase (CuZn-SOD) and selected proteases. Compared with placebo diet animals, the degree of muscle atrophy in response to immobilization was significantly reduced in astaxanthin diet animals. Further, astaxanthin supplementation significantly prevented the immobilization-induced increase in the expression of CuZn-SOD, cathepsin L, calpain, and ubiquitin in the atrophied muscle. These results support the postulate that dietary astaxanthin intake attenuates the rate of disuse muscle atrophy by inhibiting oxidative stress and proteolysis via three major proteolytic pathways.

Indolepyruvate ferredoxin oxidoreductase: An oxygen-sensitive iron-sulfur enzyme from the hyperthermophilic archaeon Thermococcus profundus.

Thermococcus profundus is a strictly anaerobic sulfur-dependent archaeon that grows optimally at 80°C by peptide fermentation. Indolepyruvate ferredoxin oxidoreductase (IOR), an enzyme involved in the peptide fermentation pathway, was purified to homogeneity from the archaeon under strictly anaerobic conditions. The maximal activity was obtained above the boiling temperature of water (105°C), with a half-life of 62min at 100°C and 20min at 105°C. IOR was oxygen-sensitive with a half-life of 7h at 25°C under aerobic conditions. The specific activity of T. profundus IOR was found to be dependent on the number of [4Fe-4S] clusters in the enzyme.

ISC-like [2Fe-2S] ferredoxin (FdxB) dimer from Pseudomonas putida JCM 20004: structural and electron-nuclear double resonance characterization.

The crystal structure of the ISC-like [2Fe-2S] ferredoxin (FdxB), probably involved in the de novo iron-sulfur cluster biosynthesis (ISC) system of Pseudomonas putida JCM 20004, was determined at 1.90-Å resolution and displayed a novel tail-to-tail dimeric form. P. putida FdxB lacks the consensus free cysteine usually present near the cluster of ISC-like ferredoxins, indicating its primarily electron transfer role in the iron-sulfur cluster. Orientation-selective electron-nuclear double resonance spectroscopic analysis of reduced FdxB in conjunction with the crystal structure has identified the innermost Fe2 site with a high positive spin population as the nonreducible iron retaining the Fe(3+) valence and the outermost Fe1 site as the reduced iron with a low negative spin density. The average g (max) direction is skewed, forming an angle of about 27.3° (±4°) with the normal of the [2Fe-2S] plane, whereas the g (int) and g (min) directions are distributed in the cluster plane, presumably tilted by the same angle with respect to this plane. These results are related to those for other [2Fe-2S] proteins in different electron transport chains (e.g. adrenodoxin) and suggest a significant distortion of the electronic structure of the reduced [2Fe-2S] cluster under the influence of the protein environment around each iron site in general.

Cyanidioschyzon merolae ferredoxin: a high resolution crystal structure analysis and its thermal stability.

Cyanidioschyzon merolae (Cm) is a single-cell red alga that grows under moderately thermophilic (40-50°C), acidic (pH 1-3) conditions. We purified a Cm ferredoxin (Fd) that was characterized as a plant-type [2Fe-2S] Fd by physicochemical techniques. X-ray crystallography revealed that the overall three-dimensional structure of CmFd was highly similar to, but slightly different from, the [2Fe-2S] Fd from Spinacia oleracea, whose growth temperature is 15-20°C. Therefore, slight structural differences, including non-covalent-bond number and amino acid sequence, may underlie the differential thermostabilities of the plant-type Fds.

Glyceraldehyde-3-phosphate dehydrogenase interacts with phosphorylated Akt resulting from increased blood glucose in rat cardiac muscle.

Here we describe the interaction of phosphorylated approximately 40 kDa protein with phosphorylated Akt which is a serine/threonine kinase resulting from increased blood glucose in rat cardiac muscle. Mass spectrometry analysis revealed that this protein was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, increase in Akt and GAPDH phosporylation and induction of their association were both observed after insulin stimulation in the H9c2 cell line derived from embryonic rat ventricle. Moreover, the activation of GAPDH was upregulated when the GAPDH phosphorylation was increased. Our data suggest that GAPDH phosphorylation and association with Akt by insulin treatment have some bearing on the enhancement of GAPDH activity.

Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei.

The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis-Menten kinetics (K(m) of 338microM and V(max) of 601micromol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1.

Enzymatic and electron paramagnetic resonance studies of anabolic pyruvate synthesis by pyruvate: ferredoxin oxidoreductase from Hydrogenobacter thermophilus.

Pyruvate: ferredoxin oxidoreductase (POR; EC 1.2.7.1) catalyzes the thiamine pyrophosphate-dependent oxidative decarboxylation of pyruvate to form acetyl-CoA and CO(2). The thermophilic, obligate chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates CO(2) via the reductive tricarboxylic acid cycle. In this cycle, POR acts as pyruvate synthase catalyzing the reverse reaction (i.e. reductive carboxylation of acetyl-CoA) to form pyruvate. The pyruvate synthesis reaction catalyzed by POR is an energetically unfavorable reaction and requires a strong reductant. Moreover, the reducing equivalents must be supplied via its physiological electron mediator, a small iron-sulfur protein ferredoxin. Therefore, the reaction is difficult to demonstrate in vitro and the reaction mechanism has been poorly understood. In the present study, we coupled the decarboxylation of 2-oxoglutarate catalyzed by 2-oxoglutarate: ferredoxin oxidoreductase (EC 1.2.7.3), which generates sufficiently low-potential electrons to reduce ferredoxin, to drive the energy-demanding pyruvate synthesis by POR. We demonstrate that H. thermophilus POR catalyzes pyruvate synthesis from acetyl-CoA and CO(2), confirming the operation of the reductive tricarboxylic acid cycle in this bacterium. We also measured the electron paramagnetic resonance spectra of the POR intermediates in both the forward and reverse reactions, and demonstrate the intermediacy of a 2-(1-hydroxyethyl)- or 2-(1-hydroxyethylidene)-thiamine pyrophosphate radical in both reactions. The reaction mechanism of the reductive carboxylation of acetyl-CoA is also discussed.

Continuous-wave and pulsed EPR characterization of the [2Fe-2S](Cys)3(His)1 cluster in rat MitoNEET.

CW EPR spectra of reduced [2Fe-2S](Cys)(3)(His)(1) clusters of mammalian mitoNEET soluble domain appear to produce features resulting from the interaction of the electron spins of the two adjacent clusters, which can be explained by employing the local spin model. This model favors the reduction of the outermost iron with His87 and Cys83 ligands, which is supported by orientation-selected hyperfine sublevel correlation (HYSCORE) characterization of the uniformly (15)N-labeled mitoNEET showing one strongly coupled nitrogen from the His87 N(delta) ligand with hyperfine coupling (15)a = 8 MHz. The (14)N and (15)N HYSCORE spectra also exhibit at least two different cross-peaks located near diagonal in the (++) quadrant, with frequencies approximately 2.8 and 2.4 MHz (N2), and the other approximately 4.0 and 3.5 MHz (N1), but did not show any of the larger splitting approximately 1.1-1.4 MHz previously seen with Rieske proteins. Further analysis with partially (15)N(3)-His-labeled protein indicates that His87 N(epsilon) cross-peaks produce resolved features (N2) in the (14)N spectrum but contribute much less than weakly coupled peptide nitrogen species to the (++) quadrant in the (15)N spectrum. It is suggested that these quantitative data may be used in future functional and theoretical studies on the mammalian mitoNEET [2Fe-2S] cluster system.

Mechanistic study on the reaction of a radical SAM dehydrogenase BtrN by electron paramagnetic resonance spectroscopy.

BtrN is a radical SAM ( S-adenosyl- l-methionine) enzyme that catalyzes the oxidation of 2-deoxy- scyllo-inosamine (DOIA) into 3-amino-2,3-dideoxy- scyllo-inosose (amino-DOI) during the biosynthesis of 2-deoxystreptamine (DOS) in the butirosin producer Bacillus circulans. Recently, we have shown that BtrN catalyzes the transfer of a hydrogen atom at C-3 of DOIA to 5'-deoxyadenosine, and thus, the reaction was proposed to proceed through the hydrogen atom abstraction by the 5'-deoxyadenosyl radical. In this work, the BtrN reaction was analyzed by EPR spectroscopy. A sharp double triplet EPR signal was observed when the EPR spectrum of the enzyme reaction mixture was recorded at 50 K. The spin coupling with protons partially disappeared by reaction with [2,2- (2)H 2]DOIA, which unambiguously proved the observed signal to be a radical on C-3 of DOIA. On the other hand, the EPR spectrum of the [4Fe-4S] cluster of BtrN during the reaction showed a complex signal due to the presence of several species. Comparison of signals derived from a [4Fe-4S] center of BtrN incubated with various combinations of products (5'-deoxyadenosine, l-methionine, and amino-DOI) and substrates (SAM and DOIA) indicated that the EPR signals observed during the reaction were derived from free BtrN, a BtrN-SAM complex, and a BtrN-SAM-DOIA complex. Significant changes in the EPR signals upon binding of SAM and DOIA suggest the close interaction of both substrates with the [4Fe-4S] cluster.

Crystallization and preliminary X-ray diffraction studies of the ISC-like [2Fe-2S] ferredoxin (FdxB) from Pseudomonas putida JCM 20004.

The iron-sulfur (Fe-S) cluster-biosynthesis (ISC) system of the gamma-proteobacterium Pseudomonas putida JCM 20004 contains a constitutively expressed vertebrate-type [2Fe-2S] ferredoxin, FdxB, which lacks the conserved free cysteine residue near the Fe-S cluster site that has been proposed to function in the catalysis of biological Fe-S cluster assembly in other bacterial homologues. Recombinant FdxB was heterologously overproduced in Escherichia coli, purified and crystallized in its oxidized form by the hanging-drop vapour-diffusion and streak-seeding methods using 1.6 M trisodium citrate dihydrate pH 6.5. The thin needle-shaped crystals diffract to 1.90 A resolution and belong to the hexagonal space group P6(1)22, with unit-cell parameters a = 87.58, c = 73.14 A. The asymmetric unit contains one protein molecule.

Characterization and mechanistic study of a radical SAM dehydrogenase in the biosynthesis of butirosin.

BtrN encoded in the butirosin biosynthetic gene cluster possesses a CXXXCXXC motif conserved within the radical S-adenosyl methionine (SAM) superfamily. Its gene disruption in the butirosin producer Bacillus circulans caused the interruption of the biosynthetic pathway between 2-deoxy-scyllo-inosamine (DOIA) and 2-deoxystreptamine (DOS). Further, in vitro assay of the overexpressed enzyme revealed that BtrN catalyzed the oxidation of DOIA under the strictly anaerobic conditions along with consumption of an equimolar amount of SAM to produce 5'-deoxyadenosine, methionine, and 3-amino-2,3-dideoxy-scyllo-inosose (amino-DOI). Kinetic analysis showed substrate inhibition by DOIA but not by SAM, which suggests that the reaction is the Ordered Bi Ter mechanism and that SAM is the first substrate and DOIA is the second. The BtrN reaction with [3-2H]DOIA generated nonlabeled, monodeuterated and dideuterated 5'-deoxyadenosines, while no deuterium was incorporated by incubation of nonlabeled DOIA in the deuterium oxide buffer. These results indicated that the hydrogen atom at C-3 of DOIA was directly transferred to 5'-deoxyadenosine to give the radical intermediate of DOIA. Generation of nonlabeled and dideuterated 5'-deoxyadenosines proved the reversibility of the hydrogen abstraction step. The present study suggests that BtrN is an unusual radical SAM dehydrogenase catalyzing the oxidation of the hydroxyl group by a radical mechanism. This is the first report of the mechanistic study on the oxidation of a hydroxyl group by a radical SAM enzyme.

MmcBC in Pelotomaculum thermopropionicum represents a novel group of prokaryotic fumarases.

The overall amino-acid sequence of MmcBC in Pelotomaculum thermopropionicum was substantially homologous (33%) to fumarase A in Escherichia coli, although its possible subunit structure was different from known fumarases and it lacked the fumarate-lyase signature sequence. Here, MmcBC in E. coli is expressed and characterized. The purified enzyme catalyzed reversible conversion of fumarate to L-malate at an optimum temperature of 70 degrees C. Its molecular size was 64.2 kDa, indicating that it consisted of one MmcB and one MmcC. EPR spectra revealed that it had an oxygen-sensitive [4Fe-4S] cluster. We propose that MmcBC represents a novel group of prokaryotic fumarases.

Crystallization and preliminary X-ray diffraction studies of a hyperthermophilic Rieske protein variant (SDX-triple) with an engineered rubredoxin-like mononuclear iron site.

In place of the Rieske [2Fe-2S] cluster, an archetypal mononuclear iron site has rationally been designed into a hyperthermophilic archaeal Rieske [2Fe-2S] protein (sulredoxin) from Sulfolobus tokodaii by three residue replacements with reference to the Pyrococcus furiosus rubredoxin sequence. The resulting sulredoxin variant, SDX-triple (H44I/A45C/H64C), has been purified and crystallized by the hanging-drop vapour-diffusion method using 65%(v/v) 2-methyl-2,4-pentanediol, 0.025 M citric acid and 0.075 M sodium acetate trihydrate pH 4.3. The crystals diffract to 1.63 A resolution and belong to the triclinic space group P1, with unit-cell parameters a = 43.56, b = 76.54, c = 80.28 A, alpha = 88.12, beta = 78.82, gamma = 73.46 degrees. The asymmetric unit contains eight protein molecules.

Identification of variant molecules of Bacillus thermoproteolyticus ferredoxin: crystal structure reveals bound coenzyme A and an unexpected [3Fe-4S] cluster associated with a canonical [4Fe-4S] ligand motif.

During the purification of recombinant Bacillus thermoproteolyticus ferredoxin (BtFd) from Escherichia coli, we have noted that some Fe-S proteins were produced in relatively small amounts compared to the originally identified BtFd carrying a [4Fe-4S] cluster. These variants could be purified into three Fe-S protein components (designated as V-I, V-II, and V-III) by standard chromatography procedures. UV-vis and EPR spectroscopic analyses indicated that each of these variants accommodates a [3Fe-4S] cluster. From mass spectrometric and protein sequence analyses together with native and SDS gel electrophoresis, we established that V-I and V-II contain the polypeptide of BtFd associated with acyl carrier protein (ACP) and with coenzyme A (CoA), respectively, and that V-III is a BtFd dimer linked by a disulfide bond. The crystal structure of the BtFd-CoA complex (V-II) determined at 1.6 A resolution revealed that each of the four complexes in the crystallographic asymmetric unit possesses a [3Fe-4S] cluster that is coordinated by Cys(11), Cys(17), and Cys(61). The polypeptide chain of each complex is superimposable onto that of the original [4Fe-4S] BtFd except for the segment containing Cys(14), the fourth ligand to the [4Fe-4S] cluster of BtFd. In the variant molecules, the side chain of Cys(14) is rotated away to the molecular surface, forming a disulfide bond with the terminal sulfhydryl group of CoA. This covalent modification may have occurred in vivo, thereby preventing the assembly of the [4Fe-4S] cluster as observed previously for Desulfovibrio gigas ferredoxin. Possibilities concerning how the variant molecules are formed in the cell are discussed.

Two tandemly arranged ferredoxin genes in the Hydrogenobacter thermophilus genome: comparative characterization of the recombinant [4Fe-4S] ferredoxins.

A thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. A gene encoding a ferredoxin involved in this cycle as an electron donor (HtFd1) was cloned and sequenced. Interestingly, another ferredoxin gene (encoding HtFd2) was found in tandem with the HtFd1 gene. These two ferredoxin genes overlapped by four bp, and transcriptional analysis revealed that they are co-transcribed as an operon. The deduced amino acid sequences of HtFd1 and HtFd2 were 42.9% identical and each contained four cysteine residues that serve as probable ligands to an iron-sulfur cluster. Spectroscopic analyses of the purified recombinant ferredoxins heterologously expressed in Escherichia coli indicated that each ferredoxin contains a single [4Fe-4S]2+/1+ cluster.

Thermococcus profundus 2-ketoisovalerate ferredoxin oxidoreductase, a key enzyme in the archaeal energy-producing amino acid metabolic pathway.

2-Ketoisovalerate ferredoxin oxidoreductase (VOR) is a key enzyme in hyperthermophiles catalyzing the coenzyme A-dependent oxidative decarboxylation of mainly aliphatic amino acid-derived 2-keto acids. The very oxygen-labile enzyme purified under anaerobic conditions from a hyperthermophilic archaeon, Thermococcus profundus, is a hetero-octamer (alphabetagammadelta)(2) consisting of four different subunits, alpha = 45,000, beta = 31,000, gamma = 22,000 and delta = 13,000, respectively. Electron paramagnetic resonance and resonance Raman spectra of the purified enzyme indicate the presence of approximately three [4Fe-4S] clusters per alphabetagammadelta-protomer, although one of the clusters has a tendency to be converted to a [3Fe-4S] form during purification. The optimal temperature for the enzyme activity is 93 +/- 2 degrees C and the cognate [4Fe-4S] ferredoxin serves as an electron acceptor of the enzyme. The purified enzyme is highly oxygen-labile (t(1/2), approximately 5 min at 25 degrees C), and is partly protected in the presence of magnesium ions, thiamine pyrophosphate and sodium chloride (t(1/2), approximately 25 min at 25 degrees C).

Follicular epithelial cell apoptosis of atretic follicles within developing ovaries of the mosquito Culex pipiens pallens.

Follicular atresia, the degeneration of developing follicles, is always incident to normal oogenesis in both oviparous and viviparous animals. Photo- and electron-microscopic observation of degenerating follicles within developing ovaries taken from blood-fed Culex pipiens pallens mosquitoes showed gradual degradation of the internal structures including yolk granules in the oocyte. The epithelial cells, which sometimes incorporated yolk granules from the oocyte along with the shrinkage of the follicle, gradually lost their uniform columnar shape, while their integrity as a covering layer remained. In situ active caspase analysis detected active enzymes in these epithelial regions. In the latest stages of atresia where either the nurse cells or oocyte were lost, the follicle was mainly comprised of irregularly shaped epithelial cells, and some of these cells' nuclei contained condensed chromatin peripherally, one of the characteristics of apoptotic cells. Also terminaldeoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling treatment indicated that DNA fragmentation occurred in these follicles. It seems likely that in atretic follicles the epithelial cells survive to play key roles in the event, and then finally undergo their own apoptotic cell death so as to give the developmental site to the next follicle in the same ovariole.