NO reductase from Bacillus azotoformans is a bifunctional enzyme accepting electrons from menaquinol and a specific endogenous membrane-bound cytochrome c551

Bacillus azotoformans is a Gram-positive denitrifying soil bacterium, which is capable of respiring nitrate, nitrite, nitric oxide, and nitrous oxide under anaerobic conditions. It contains a unique menaquinol-dependent nitric oxide reductase (qCu(A)NOR) with a Cu(A) center in its small subunit. The qCu(A)NOR exhibits menaquinol-dependent NO reductase activity, whereas reduced horse heart cytochrome c was inactive. Here we describe the purification of three membrane-bound c cytochromes from B. azotoformans. Their apparent molecular masses on SDS-PAGE are approximately 11 kDa. At neutral pH, these c cytochromes are negatively charged and the E(m) for all is close to 150 mV. Only one of these c cytochromes, which exhibits an alpha-band maximum at 551 nm, acts as a direct electron donor to qCu(A)NOR. Further investigation demonstrated that this cytochrome c(551) possesses two lipoyl moieties, which presumably function to anchor it to the membrane. Steady-state kinetic studies reveal that cytochrome c(551) is a noncompetitive inhibitor of NO reduction when menaquinol is used as an electron donor. This finding points to the presence of two different electron donation pathways in qCu(A)NOR. The ability of qCu(A)NOR to accept electrons from both menaquinol and cytochrome c(551) might be related to the regulation of the rate of NO reduction especially as a defense mechanism of B. azotoformans against the toxicity of NO. Growth experiments in batch culture indeed show that B. azotoformans is highly NO tolerant, in contrast to, for example, Paracoccus denitrificans that has a monofunctional cytochrome c-dependent NOR. We propose that the menaquinol pathway, which has a 4-fold greater maximal activity than the pathway via cytochrome c(551), is used for NO detoxification, whereas electron donation via the endogenous cytochrome c involves the cytochrome b(6)f complex serving the bioenergetic needs of the organism.     

Quantitative histochemistry of three mouse hind-limb muscles: the relationship between calcium-stimulated myofibrillar ATPase and succinate dehydrogenase activities

Summary A quantitative modification of Meijer's calcium-lead capture method, for the demonstration of calcium-stimulated myofibrillar ATPase activity at physiological pH, is described. A range of myofibrillar ATPase activities has been found among fast muscle fibres in two mouse hind-limb muscles. The myofibrillar ATPase activity of fast muscle fibres is 1.5–3 times higher than the myofibrillar ATPase activity of slow muscle fibres.Myofibrillar ATPase activities and succinate dehydrogenase activities of individual muscle fibres have been determined in serial sections. Activities of the two enzymes are correlated positively in soleus (fast and slow fibres), and negatively in plantaris (almost all fast) and extensor digitorum longus muscle (all fast). However, this correlation is not significant among the oxidative fibres in the extensor digitorum longus. The fibres of the latter muscle cannot be classified satisfactorily into two sub-types.     

Distribution of leptocephali of the freshwater eels,genus <Emphasis Type="Italic">Anguilla</Emphasis>, in the waters off west Sumatra in the Indian Ocean

A research cruise was conducted in the eastern Indian Ocean off west Sumatra, Indonesia, in June 2003 to learn about the spawning and larval ecology of the tropical freshwater eels of the genus Anguilla in the region. A total of 43 anguillid leptocephali were collected during the cruise and they were genetically identified as 41 Anguilla bicolor bicolor, 1 Anguilla marmorata, and 1 Anguilla interioris. A. bicolor bicolor leptocephali were 44.1–55.5 mm TL and most of them were at the fully grown stage. Reexamination of the historical data of Jespersen (1942) also suggested a relatively low abundance of small size leptocephali (<40 mm) of this species off west Sumatra. Although the study area has long been considered to be a spawning site of A. bicolor bicolor, the distributions of leptocephali from the two surveys and the patterns of ocean currents in the region suggest the possibility that the main spawning area of this species is located farther offshore.     

Structural and biochemical characterization of flavoredoxin from the archaeon Methanosarcina acetivorans

Flavoredoxin is a FMN-containing electron transfer protein that functions in the energy-yielding metabolism of Desulfovibrio gigas of the Bacteria domain. Although characterization of this flavoredoxin is the only one reported, a database search revealed homologues widely distributed in both the Bacteria and Archaea domains that define a novel family. To improve our understanding of this family, a flavoredoxin from Methanosarcina acetivorans of the Archaea domain was produced in Escherichia coli and biochemically characterized, and a high-resolution crystal structure was determined. The protein was shown to be a homodimer with a subunit molecular mass of 21 kDa containing one noncovalently bound FMN per monomer. Redox titration showed an E(m) of -271 mV with two electrons, consistent with no semiquinone observed in the potential range studied, a result suggesting the flavoredoxin functions as a two-electron carrier. However, neither of the obligate two-electron carriers, NAD(P)H and coenzyme F420H2, was a competent electron donor, whereas 2[4Fe-4S] ferredoxin reduced the flavoredoxin. The X-ray crystal structure determined at 2.05 A resolution revealed a homodimer containing one FMN per monomer, consistent with the biochemical characterization. The isoalloxazine ring of FMN was shown buried within a narrow groove approximately 10 A from the positively charged protein surface that possibly facilitates interaction with the negatively charged ferredoxin. The structure provides a basis for predicting the mechanism by which electrons are transferred between ferredoxin and FMN. The FMN is bound with hydrogen bonds to the isoalloxazine ring and electrostatic interactions with the phosphate moiety that, together with sequence analyses of homologues, indicate a novel FMN binding motif for the flavoredoxin family.     

Characterization of the RnfB and RnfG Subunits of the Rnf Complex from the Archaeon Methanosarcina acetivorans

Rnf complexes are redox-driven ion pumps identified in diverse species from the domains Bacteria and Archaea, biochemical characterizations of which are reported for two species from the domain Bacteria. Here, we present characterizations of the redox-active subunits RnfG and RnfB from the Rnf complex of Methanosarcina acetivorans, an acetate-utilizing methane-producing species from the domain Archaea. The purified RnfG subunit produced in Escherichia coli fluoresced in SDS-PAGE gels under UV illumination and showed a UV-visible spectrum typical of flavoproteins. The Thr166Gly variant of RnfG was colorless and failed to fluoresce under UV illumination confirming a role for Thr166 in binding FMN. Redox titration of holo-RnfG revealed a midpoint potential of −129 mV for FMN with n = 2. The overproduced RnfG was primarily localized to the membrane of E. coli and the sequence contained a transmembrane helix. A topological analysis combining reporter protein fusion and computer predictions indicated that the C-terminal domain containing FMN is located on the outer aspect of the cytoplasmic membrane. The purified RnfB subunit produced in E. coli showed a UV-visible spectrum typical of iron-sulfur proteins. The EPR spectra of reduced RnfB featured a broad spectral shape with g values (2.06, 1.94, 1.90, 1.88) characteristic of magnetically coupled 3Fe-4S and 4Fe-4S clusters in close agreement with the iron and acid-labile sulfur content. The ferredoxin specific to the aceticlastic pathway served as an electron donor to RnfB suggesting this subunit is the entry point of electrons to the Rnf complex. The results advance an understanding of the organization and biochemical properties of the Rnf complex and lay a foundation for further understanding the overall mechanism in the pathway of methane formation from acetate.     

Cytokine patterns during dengue shock syndrome

OBJECTIVE: To investigate the patterns of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, interferon-gamma (IFN-gamma) and interleukin-1 receptor antagonist (IL-1Ra) during the course of dengue shock syndrome. DESIGN: Prospective clinical study. SETTING: Pediatric Intensive Care Unit, Dr. Kariadi Hospital, the university hospital of Diponegoro University, Semarang, Indonesia. PATIENTS: Fifty children with dengue shock syndrome. MEASUREMENTS: The plasma concentration and the ex vivo production, with and without lipopolysaccharide (LPS), of TNF-alpha, IL-1beta and IL-1Ra were measured in duplicate by nonequilibrium radioimmunoassay (RIA); IFN-gamma and IL-6 were measured by ELISA. RESULTS: During the acute phase, the plasma concentrations and the ex vivo production without LPS of IL-1Ra were considerably elevated and returned to normal on recovery. However, the ex vivo LPS-stimulated production of the proinflammatory cytokines TNF-alpha and IL-1beta were considerably depressed. Also, these concentrations returned towards normal on recovery. In non-survivors, the plasma concentrations of IL-6 and IL-1Ra were significantly higher than in survivors (p = 0.00001 and p = 0.0005, respectively). In addition, the ex vivo production of IL-1Ra in non-survivors was significantly higher than in survivors, both without LPS stimulation (p = 0.0008) and with LPS (p < 0.004). IL-1Ra was significantly associated with mortality (p = 0.007). CONCLUSION: Since IL-1Ra was significantly associated with mortality, this measurement may be used as an index of disease severity in dengue shock syndrome.     

Strengthening social capital for propelling collective action in mangrove management

The concept of social capital has gained lots of attention as an important instrument to induce collective action on Common Pool Resources management. However, evidence demonstrated amply that social capital alone was not always enough to encourage collective action. There were other factors needed as a leverage to activate social capital but research regarding this issue is still limited. This research was intended to elucidate how to strengthen the role of social capital and the preconditions required to encourage community members to conduct collective action. The research was carried out using survey methods at the eastern coastal area of East Sinjai sub district, South Sulawesi, Indonesia. The research results show that strong social capital, indicated by high value score of trust, norm and networking would not always engender collective action in natural resource management. In order to achieve collective action, social capital had to be activated to function optimally. This was done through the intervention of symbolic power which is inherent in role models to initiate and mobilize action in mangrove management. The process to convince people to perform collective action was a crucial one which had to be resolved and is known as common knowledge. External support from local governments could facilitate the emergence of symbolic power through provision of enabling conditions for leadership promotion.     

A novel copper A containing menaquinol NO reductase from Bacillus azotoformans

The molecular biology and biochemistry of denitrification in gram-negative bacteria has been studied extensively. However, little is known about this process in gram-positive bacteria. We have purified the NO reductase from the cytoplasmic membrane of the gram-positive bacterium Bacillus azotoformans. The purified enzyme consists of two subunits with apparent molecular masses of 16 and 40 kDa based on SDS-PAGE. Analytical and spectroscopic determinations revealed the presence of one non-heme iron, two copper atoms and of two b-type hemes per enzyme complex. Heme c was absent. Using EPR and UV-visible spectroscopy, it was determined that one of the hemes is a low-spin heme b, in which the two axial histidine imidazole planes are positioned at an angle of 60-70 degrees. The second heme b is high-spin binding CO in the reduced state. The high-spin heme center and the non-heme iron are EPR silent. They are proposed to form a binuclear center where reduction of NO occurs. There are two novel features of this enzyme that distinguish it from other NO reductases. First, the enzyme contains copper in form of copper A, an electron carrier up to now only detected in cytochrome oxidases and nitrous oxide reductases. Second, the enzyme uses menaquinol as electron donor, whereas cytochrome c, which is the substrate of other NO reductases, is not used. Copper A and both hemes are reducible by menaquinol. This new NO reductase is thus a menaquinol:NO oxidoreductase. With respect to its prosthetic groups the B. azotoformans NO reductase is a true hybrid between copper A containing cytochrome oxidases and NO reductases present in gram-negative bacteria. It may represent the most ancient "omnipotent" progenitor of the family of heme-copper oxidases.     

Gonadal development in a giant threatened reef fish,the humphead wrasse Cheilinus undulatus,and its relationship to international trade

An opportunity arose to obtain humphead wrasse Cheilinus undulatus specimens between 2006 and 2009 from Indonesia, the major source and exporting country of this species, making study on its early gonad development possible for the first time. Protogynous hermaphroditism, previously proposed for this species, was confirmed in this study. Based on histological examination of 178 specimens, mainly <500 mm total length (L_T) and ranging from 208 to 1290 mm L_T (119·1 g to 43·0 kg whole body mass), the minimum body sizes for female and male sexual maturation were determined to be 650 and 845 mm L_T, respectively. Primary male development through juvenile sexual differentiation was not detected. A unique blind pouch, with a possible sperm storage function and associated with the testis, was reported for the first time in the Labridae. In Hong Kong retail markets, the global trading centre for this valuable species, live C. undulatus on sale for food were dominated by body sizes <500 mm L_T between 1995 and 2009, reflecting an international trade largely focused on juveniles. In consideration of these findings, and given the threatened status of this species, management for C. undulatus capture and trade nationally and internationally are discussed with recommendations for ensuring sufficient spawning biomass in exploited populations and for sustainable trade.     

Relationship between myoglobin and succinate dehydrogenase in mouse soleus and plantaris muscle fibres

Summary This report describes a quantitative histochemical study of myoglobin in skeletal muscle fibres. The muscle fibres were classified as fast or slow on the basis of their quantitative myofibrillar ATPase histochemistry. A large range of myoglobin absorbance values was found among fast skeletal muscle fibres. This range was relatively small among slow fibres. The concentrations of myoglobin and the activities of succinate dehydrogenase in individual muscle fibres in serial sections are weakly correlated in both the mouse soleus and plantaris muscle. The myoglobin concentration is higher in fast and slow oxidative soleus muscle fibres and the succinate dehydrogenase activity in these fibres is lower than in oxidative plantaris muscle fibres in the same range of cross-sectional area.