Further investigation on the turnover of Escherichia coli biotin synthase with dethiobiotin and 9-mercaptodethiobiotin as substrates

Biotin synthase, a member of the "radical-SAM" family, produces biotin by inserting a sulfur atom between C-6 and C-9 of dethiobiotin. Each of the two saturated carbon atoms is activated through homolytic cleavage of a C-H bond by a deoxyadenosyl radical, issued from the monoelectronic reduction of S-adenosylmethionine (SAM or AdoMet). An important unexplained observation is that the enzyme produces only 1 mol of biotin per enzyme monomer. Some possible reasons for this absence of multiple turnovers are considered here, in connection with the postulated mechanisms. There is a general agreement among several groups that the active form of biotin synthase contains one (4Fe-4S)(2+,1+) center, which mediates the electron transfer to AdoMet, and one (2Fe-2S)(2+) center, which is considered the sulfur source [Ugulava, N. B., Sacanell, C. J., and Jarrett, J. T. (2001) Biochemistry 40, 8352-8358; Tse Sum Bui, B., Benda, R., Schunemann, V., Florentin, D., Trautwein, A. X., and Marquet, A. (2003) Biochemistry 42, 8791-8798; Jameson, G. N. L., Cosper, M. M., Hernandez, H. L., Johnson, M. K., and Huynh, B. H. (2004) Biochemistry 43, 2022-2031]. An alternative hypothesis considers that biotin synthase has a pyridoxal phosphate (PLP)-dependent cysteine desulfurase activity, producing a persulfide which could be the sulfur donor. The absence of turnover was explained by the inhibition due to deoxyadenosine, an end product of the reaction [Ollagnier-de Choudens, S., Mulliez, E., and Fontecave, M. (2002) FEBS Lett. 535, 465-468]. In this work, we show that our purified enzyme has no cysteine desulfurase activity and the required sulfide has to be added as Na(2)S. It cannot be replaced by cysteine, and consistently, PLP has no effect. We observed that deoxyadenosine does not inhibit the reaction either. On the other hand, if the (2Fe-2S)(2+) center is the sulfur source, its depletion after reaction could explain the absence of turnover. We found that after addition of fresh cofactors, including Fe(2+) and S(2)(-), either to the assay when one turn is completed or after purification of the reacted enzyme by different techniques, only a small amount of biotin (0.3-0.4 equiv/monomer) is further produced. This proves that an active enzyme cannot be fully reconstituted after one turn. When 9-mercaptodethiobiotin, which already contains the sulfur atom of biotin, is used as the substrate, the same turnover of one is observed, with similar reaction rates. We postulate that the same intermediate involving the (2Fe-2S) cluster is formed from both substrates, with a rate-determining step following the formation of this intermediate.     

Depth selection and life history strategies as mutually exclusive responses to risk of fish predation in <Emphasis Type="Italic">Daphnia</Emphasis>

We tested if pelagic crustaceans of the genus Daphnia use different anti-predator defences in environmental conditions that do or do not offer deep refuge from planktivorous fish. We kept Daphnia catawba in two series of 9-m deep enclosures with and without caged cyprinid fish Phoxinus eos. In one series of enclosures, Daphnia could select its depth of residence and hide in deep dark water layers to avoid anticipated fish predation, while in another series of enclosures, a plankton net barrier fixed at 2-m depth forced them to stay in subsurface zone exposed to fish kairomones. We compared depth residence and migratory behaviour strategies with life history strategies (body size and size at first reproduction, diapause induction) in Daphnia exposed or not to fish kairomones with or without deep refuge. In deep enclosures with fish, Daphnia spent daytime hours in deep dark layers while at night, they resided closer to the water surface. Yet, no change in life history parameters of migrating individuals was observed compared to the fish-free conditions. In enclosures with fish, where the net barrier forced Daphnia to reside in subsurface zone, they produced smaller offspring, matured at smaller size and achieved lower maximum body length compared to the fish-free conditions. However, they did not produce diapausing eggs. Our experimental study supports the hypothesis that diel vertical migration behaviour with daytime residence in deep, dark water are the preferred antipredator strategy chosen by Daphnia facing anticipated fish predation over life history changes such as reduced size and low growth rate which are used when dark deep refuge is not present or accessible.     

A matrix metalloproteinase mediates airway remodeling in Drosophila

Organ size typically increases dramatically during juvenile growth. This growth presents a fundamental tension, as organs need resiliency to resist stresses while still maintaining plasticity to accommodate growth. The extracellular matrix (ECM) is central to providing resiliency, but how ECM is remodeled to accommodate growth is poorly understood. We investigated remodeling of Drosophila respiratory tubes (tracheae) that elongate continually during larval growth, despite being lined with a rigid cuticular ECM. Cuticle is initially deposited with a characteristic pattern of repeating ridges and valleys known as taenidia. We find that for tubes to elongate, the extracellular protease Mmp1 is required for expansion of ECM between the taenidial ridges during each intermolt period. Mmp1 protein localizes in periodically spaced puncta that are in register with the taenidial spacing. Mmp1 also degrades old cuticle at molts, promotes apical membrane expansion in larval tracheae, and promotes tube elongation in embryonic tracheae. Whereas work in other developmental systems has demonstrated that MMPs are required for axial elongation occurring in localized growth zones, this study demonstrates that MMPs can also mediate interstitial matrix remodeling during growth of an organ system.     

Normal aging and cognition

It is now well documented that normal aging modifies the cognitive functioning and most observations suggest that cognition evolves in the direction of deterioration. The more frequently impaired functions are memory, attention and visual-spatial abilities. On the other hand, some abilities seem to increase, such as vocabulary. Considering the aging effect on cognition, questions remain regarding directionality, universality and reversibility. A great variability in aged related impacts is observed among subjects and among cognitive domains. Some individuals evolved more rapidly than others. Some cognitive functions are more affected by aging than others. General and specific factors are hypothesized to explain the aged related cognitive decline. Among them, educational level, health, cognitive style, life style, personality, are likely to modulate the aged related cognitive evolution by influencing attentional resources and cerebral plasticity. Cognitive resources are essential to develop adaptative strategies. During the life span, resources are activated and increased by learning and training. Considering the role of cognitive resources, successful aging is dependent on several conditions : absence of disease leading to a loss of autonomy, maintenance of cognitive and physical activities, and active and social engaged lifestyle.     

Protein expression pattern in experimental pneumococcal meningitis

In this study, we investigated cytokine expression during experimental pneumococcal meningitis. Mice were intracisternally infected with Streptococcus pneumoniae and treated with ceftriaxone starting at 24 h after infection. At different time points before and after antibiotic therapy, the cytokine expression pattern was determined in mouse brains using protein arrays. Underlining the power of this method, the meningitis-relevant cytokines interleukin-1beta (IL-1beta), IL-6, KC, macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractant protein-1 (MCP-1/CCL2) were markedly elevated in infected animals. Newly identified proteins during the acute stage of the disease (until 30 h after infection) included lymphotactin (XCL-1), MIP-1gamma (CCL9) and MCP-5 (CCL12), cytokine responsive gene- 2 (CRG-2/CXCL10) and CXCL16, and insulin-like growth factor binding protein 3 (IGFBP3). During later stages, an induction of T-cell activation-3 (TCA-3/CCL1), platelet factor-4 (PF-4/CXCL4) and stromal derived factor-1alpha (SDF-1alpha/CXCL13), and IL-4 was observed. The validity of this method was supported by an additional ELISA analysis of the expression profile of CXCL16 and IGFBP3, which was identical to that observed by protein array. In conclusion, the use of protein array technology led to an extension of the current picture of protein expression in pneumococcal meningitis. Most important, new factors that might play a role in pneumococcal meningitis were identified.     

An overview of fungal community diversity in diseased hop plantations

Samples were taken from several hop fields presenting various symptoms. Fifty-nine pure filamentous fungal strains were isolated and identified through genomic DNA preparations, PCR amplification of the ribosomal DNA internal transcribed spacer region and database interrogations. The most frequent genera were Alternaria (16 isolates) and Epicoccum (14 isolates). The ecosystem was shown to be very diverse, since as many as 27 species belonging to 17 genera were recovered. Furthermore, many of the isolated fungi are known to be involved in phytopathogenesis.     

Leptin inhibits swallowing in rats

Swallowing is under the control of premotoneurons located in the medullary solitary tract nucleus. Although rats with transected midbrain do not seek out food, they are able to ingest food present near the mouth, and acute food deprivation induces an increase in food intake. Leptin is a satiety signal that regulates feeding behavior. Because leptin receptors are found within the caudal brainstem, and because food intake is regulated in midbrain transected rats, this study tested the hypothesis that leptin is able to modify the activity of premotoneurons involved in swallowing. Leptin was microinjected at the subpostremal level of the medullary solitary tract nucleus in anesthetized Wistar rats. Electromyographic electrodes in sublingual muscles allowed recording of swallowing induced by stimulation of sensitive fibers of the superior laryngeal nerve. Repeated stimulation induced rhythmic swallowing. Microinjection of leptin (0.1 pg and 0.1 ng) in the swallowing center induced an inhibition of rhythmic swallowing (latency of <30 s) as shown by the reduced number and strength of electromyographic activities, which could last several minutes. The threshold of the leptin-induced inhibition was close to 0.1 pg. Interestingly, the inhibitory effect of leptin was not observed in leptin receptor-deficient Zucker rats. Here we show that, in Wistar rats, leptin already known to modulate the discharge of medullary solitary tract nucleus-sensitive neurons involved in satiety reflexes can also modify the activity of swallowing premotoneurons, thereby inhibiting an essential motor component of feeding behavior.     

Treatment of rats with calpain inhibitors prevents sepsis-induced muscle proteolysis independent of atrogin-1/MAFbx and MuRF1 expression

Muscle wasting in sepsis is a significant clinical problem because it results in muscle weakness and fatigue that may delay ambulation and increase the risk for thromboembolic and pulmonary complications. Treatments aimed at preventing or reducing muscle wasting in sepsis, therefore, may have important clinical implications. Recent studies suggest that sepsis-induced muscle proteolysis may be initiated by calpain-dependent release of myofilaments from the sarcomere, followed by ubiquitination and degradation of the myofilaments by the 26S proteasome. In the present experiments, treatment of rats with one of the calpain inhibitors calpeptin or BN82270 inhibited protein breakdown in muscles from rats made septic by cecal ligation and puncture. The inhibition of protein breakdown was not accompanied by reduced expression of the ubiquitin ligases atrogin-1/MAFbx and MuRF1, suggesting that the ubiquitin-proteasome system is regulated independent of the calpain system in septic muscle. When incubated muscles were treated in vitro with calpain inhibitor, protein breakdown rates and calpain activity were reduced, consistent with a direct effect in skeletal muscle. Additional experiments suggested that the effects of BN82270 on muscle protein breakdown may, in part, reflect inhibited cathepsin L activity, in addition to inhibited calpain activity. When cultured myoblasts were transfected with a plasmid expressing the endogenous calpain inhibitor calpastatin, the increased protein breakdown rates in dexamethasone-treated myoblasts were reduced, supporting a role of calpain activity in atrophying muscle. The present results suggest that treatment with calpain inhibitors may prevent sepsis-induced muscle wasting.     

Biotin synthase mechanism: mutagenesis of the YNHNLD conserved motif

Biotin synthase, a member of the "radical SAM" family, catalyzes the final step of the biotin biosynthetic pathway, namely, the insertion of a sulfur atom into dethiobiotin (DTB). The active form of the enzyme contains two iron-sulfur clusters, a [4Fe-4S](2+) cluster liganded by Cys-53, Cys-57, and Cys-60 and the S-adenosylmethionine (AdoMet or SAM) cosubstrate and a [2Fe-2S](2+) cluster liganded by Cys-97, Cys-128, Cys-188, and Arg-260. Single-point mutation of each of these six conserved cysteines produced inactive variants. In this work, mutants of other highly conserved residues from the Y(150)NHNLD motif are described. They have properties similar to those of the wild-type enzyme with respect to their cluster content and characteristics. For all of them, the as-isolated form, which contains an air-stable [2Fe-2S](2+) center, can additionally accommodate an air-sensitive [4Fe-4S](2+) center which is generated by incubation under anaerobic conditions with Fe(2+) and S(2-). Their spectroscopic properties are similar to those of the wild type. However, they are inactive, except the mutant H152A that exhibits a weak activity. We show that the mutants, inactive in producing biotin, are also unable to cleave AdoMet and to produce the deoxyadenosyl radical (AdoCH(2)(*)). In the case of H152A, a value of 5.5 +/- 0.4 is found for the 5'-deoxyadenosine (AdoCH(3)):biotin ratio, much higher than the value of 2.8 +/- 0.3 usually observed with the wild type. This reveals a greater contribution of the abortive process in which the AdoCH(2)(*) radical is quenched by hydrogen atoms from the protein or from some components of the system. Thus, in this case, the coupling between the production of AdoCH(2)(*) and its reaction with the hydrogen at C-6 and C-9 of DTB is less efficient than that in the wild type, probably because of geometry's perturbation within the active site.