Allosteric activation of rat liver microsomal [hydroxymethylglutaryl-CoA reductase (NADPH)]kinase by nucleoside phosphates

Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase activity is enhanced about 5 fold by 2 mM of either AMP or ADP. Activation constants, Ka, for AMP and ADP are 17 microM and 430 microM respectively, showing that AMP is a more potent activator than ADP. This property is expressed by increasing not only the rate of reductase inactivation but also the rate of reductase phosphorylation from [gamma-32P]ATP. GTP can replace ATP as substrate of reductase kinase but GMP and GDP cannot replace AMP as activators. Kinetic studies show that ATP can only act as a substrate. Nucleoside mono or diphosphates and nucleoside triphosphates, thus, appear to bind to different sites on microsomal HMG-CoA reductase kinase. Nucleoside mono or diphosphates act as allosteric activators of reductase kinase. The adenosyl moiety and the unaltered phosphate ester at the 5' position are two essential features of the activator molecule. Phosphorylation of reductase either by microsomal or cytosolic AMP-activated reductase kinase produces an 80% inactivation, with a concomitant incorporation of 0.8 mol of 32P per mol of reductase (Mr 55,000). In both cases exhaustive tryptic digestion of 32P-labeled HMG-CoA reductase, which had been denatured in 2M urea, yields two major phosphopeptides, the phosphoryl group being bound to serine residues.     

Affinity labeling of the catalytic and AMP allosteric sites of 3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase by 5'-p-fluorosulfonylbenzoyladenosine

The nucleotide analogue 5'-p-fluorosulfonylbenzoyladenosine (FSBA) reacts irreversibly with rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase kinase, causing a rapid loss of the AMP activation capacity and a slower inactivation of the catalytic activity. The rate constant for loss of AMP activation is about 10 times higher (kappa 1 = 0.112 min-1) than the rate constant of inactivation (kappa 2 = 0.0106 min-1). There is a good correspondence between the time-dependent inactivation of reductase kinase and the time-dependent incorporation of 5'-p-sulfonylbenzoyl[14C]adenosine ([14C]SBA). An average of 1.65 mol of reagent/mol of enzyme subunit is bound when reductase kinase is completely inactivated. The time-dependent incorporation is consistent with the postulate that covalent reaction of 1 mol of SBA/mol of subunit causes complete loss of AMP activation, whereas reaction of another mole of SBA/mol of subunit would lead to total inactivation. Protection against inactivation by the reagent is provided by the addition of Mg2+, AMP, Mg-ATP, or Mg-AMP to the incubation mixtures. In contrast, addition of ATP, 2'-AMP, or 3'-AMP has no effect on the rate constants. Mg-ATP protects preferentially the catalytic site against inactivation, whereas Mg-AMP at low concentration protects preferentially the allosteric site. Mg-ADP affords less protection than Mg-AMP to the allosteric site when both nucleotides are present at a concentration of 50 microM with 7.5 mM Mg2+. Experiments done with [14C]FSBA in the presence of some protectants have shown that a close correlation exists between the pattern of protection observed and the binding of [14C]SBA. The postulate is that there exists a catalytic site and an allosteric site in the reductase kinase subunit and that Mg-AMP is the main allosteric activator of the enzyme.     

Activation of rat liver cytosolic 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase by adenosine 5'-monophosphate

Inactivation of 3-hydroxy-3-methylglutaryl Coenzyme A reductase by reductase kinase and ATP-Mg needs either ADP or 5'-AMP as cofactors. 5'-AMP is a more potent activator of cytosolic reductase kinase than ADP. This capacity is expressed by increasing not only the rate of reductase inactivation, but also the rate of reductase phosphorylation from [gamma-32P]ATP. Activation constants, Ka, for 5'-AMP and ADP are 20 microM and 420 microM respectively. Neither 3'-AMP nor 2'-AMP activate reductase kinase. Other nucleoside monophosphates like UMP, CMP and GMP cannot replace 5'-AMP as activators of reductase kinase.     

Free radical production, catalase and superoxide dismutase activities and membrane integrity during senescence of petals of cut carnations (Dianthus caryophyllus)

There is a sudden increase in free radical levels, measured from the electron spin resonance (ESR) signal, in cut carnation ( Dianthus caryophyllus L. cv. Ember) petal powders between the end of blooming and the onset of withering. There is also an increase in the microsomal generation of superoxide radicals (measured from the ESR-Tiron signal). These increases correspond to a decrease of polar lipids content, a slight increase in peroxides and to the onset of a sudden efflux of electrolytes. A correlation is established between free radical production and the loss of membrane integrity. Catalase (EC 1.11.1.6) activity increases progressively until complete withering and an hypothesis concerning the action of this enzyme is proposed. The changes in superoxide dismutase (EC 1.15.1.1) activity appear to be independent of the amplitude of the ESR-Tiron signal.     

Roles of BdUNICULME4 and BdLAXATUM‐A in the non‐domesticated grass Brachypodium distachyon

In cultivated grasses, tillering, spike architecture and seed shattering represent major agronomical traits. In barley, maize and rice, the NOOT‐BOP‐COCH‐LIKE (NBCL) genes play important roles in development, especially in ligule development, tillering and flower identity. However, compared with dicots, the role of grass NBCL genes is underinvestigated. To better understand the role of grass NBCLs and to overcome any effects of domestication that might conceal their original functions, we studied TILLING nbcl mutants in the non‐domesticated grass Brachypodium distachyon. In B. distachyon, the NBCL genes BdUNICULME4 (CUL4) and BdLAXATUM‐A (LAXA) are orthologous, respectively, to the barley HvUniculme4 and HvLaxatum‐a, to the maize Zmtassels replace upper ears1 and Zmtassels replace upper ears2 and to the rice OsBLADE‐ON‐PETIOLE1 and OsBLADE‐ON‐PETIOLE2/3. In B. distachyon, our reverse genetics study shows that CUL4 is not essential for the establishment of the blade–sheath boundary but is necessary for the development of the ligule and auricles. We report that CUL4 also exerts a positive role in tillering and a negative role in spikelet meristem activity. On the other hand, we demonstrate that LAXA plays a negative role in tillering, positively participates in spikelet development and contributes to the control of floral organ number and identity. In this work, we functionally characterized two new NBCL genes in a context of non‐domesticated grass and highlighted original roles for grass NBCL genes that are related to important agronomical traits.     

Litter quality and inflammatory response are dependent on mating strategy in a reptile

Maintenance of health and the production of offspring are competing processes that can result in trade-offs. As vertebrates invest substantial resources in their immune system, it is crucial to understand the interactions between immunity and reproductive strategies. In the lizard Zootoca vivipara, females have condition- and context-dependent mating strategies. We predicted that, if the risk of infection is higher for polyandrous females, then polyandrous females should invest more in immune system while monandrous females should invest more in reproduction. In order to test our prediction, we captured 62 gravid females of known age in a natural population; we kept them until parturition to access to their offspring. Then, using microsatellite marker-based paternity analyses within litters, we determine the mating strategy of females (monandrous or polyandrous). Females were also challenged with PHA to estimate their inflammatory response. Our results show that polyandrous females have a higher PHA response than the monandrous females, and that monandrous females produce more males and more juveniles of better body condition than polyandrous females. The relationship between mating behaviour and immune function may have consequences for females and may shape the evolution of mating systems.     

Biodiversity monitoring: some proposals to adequately study species’ responses to climate change

Climate change affects all levels of biology and is a major threat for biodiversity. Hence, it is fundamental to run biodiversity monitoring programs to understand the effects of climate change on the biota and to be able to adjust management and conservation accordingly. So far, however, very few existing monitoring programs allow for the detection of climate change effects, as shown by a survey undertaken by the European project EuMon. Despite this shortcoming, several methods exist which allow to make inferences from existing data by integrating data across different monitoring programs: correlative analyses, meta-analyses and models. In addition, experiments are thought to be useful tools to understand the effects of climate change on plants and animals. Here, we evaluate the utility of these four main approaches. All these methods allow to evaluate long term effects of climate change and make predictions of species’ future development, but they are arguable. We list and compare their benefits and inconveniences, which can lead to uncertainties in the extrapolation of species responses to global climate change. Individual characteristics and population parameters have to be more frequently monitored. The potential evolution of a species should be also modelled, to extrapolate results across spatial and temporal scales as well as to analyse the combined effects of different climatic and biotic factors, including intra but also interspecific relationships. We conclude that a combination of methodologies would be the most promising tool for the assessment of biological responses to climate change, and we provide some thoughts about how to do so. Particularly, we encourage long-term studies along natural gradients (altitudinal or latitudinal) on the same species/habitats to be able to extrapolate to large geographic scales, and to have more complete data sets, necessary to understand the mechanisms of responses. Such data may provide a more accurate base for simulations across spatial and temporal scales, especially if they are publicly available in a common database. These recommendations could allow the adaptation of species management and the development of conservation tools to climate change which threatens species.     

Informed dispersal, heterogeneity in animal dispersal syndromes and the dynamics of spatially structured populations

There is accumulating evidence that individuals leave their natal area and select a breeding habitat non-randomly by relying upon information about their natal and future breeding environments. This variation in dispersal is not only based on external information (condition dependence) but also depends upon the internal state of individuals (phenotype dependence). As a consequence, not all dispersers are of the same quality or search for the same habitats. In addition, the individual's state is characterized by morphological, physiological or behavioural attributes that might themselves serve as a cue altering the habitat choice of conspecifics. These combined effects of internal and external information have the potential to generate complex movement patterns and could influence population dynamics and colonization processes. Here, we highlight three particular processes that link condition-dependent dispersal, phenotype-dependent dispersal and habitat choice strategies: (1) the relationship between the cause of departure and the dispersers' phenotype; (2) the relationship between the cause of departure and the settlement behaviour and (3) the concept of informed dispersal, where individuals gather and transfer information before and during their movements through the landscape. We review the empirical evidence for these processes with a special emphasis on vertebrate and arthropod model systems, and present case studies that have quantified the impacts of these processes on spatially structured population dynamics. We also discuss recent literature providing strong evidence that individual variation in dispersal has an important impact on both reinforcement and colonization success and therefore must be taken into account when predicting ecological responses to global warming and habitat fragmentation.     

Female choice for heterozygous mates changes along successive matings in a lizard

Female mate choice and female multiple mating are major focuses of studies on sexual selection. In a multiple mating context, the benefits of mate choice can change along successive matings, and female choice would be expected to change accordingly. We investigated sequential female mate choice in the moderately polyandrous common lizard (Zootoca vivipara, synonym Lacerta vivipara). Along successive mating opportunities, we found that females were relatively unselective for the first mate, but accepted males of higher heterozygosity for subsequent mating, consistent with the trade-up choice hypothesis. We discuss the evidence of trade-up mate choice in squamates and generally trade-up for mate heterozygosity in order to motivate new studies to fill gaps on these questions.     

Optimized culture conditions for the production of furanocoumarins by micropropagated shoots of Ruta graveolens

Ruta graveolens in vitro cultures are a potential source of secondary metabolites (furanocoumarins) of significant medical interest. Experiments led in our laboratory showed that micropropagated shoots were richer in furanocoumarins than any other plant material. In order to optimize the molecule production by such cultivation systems, several factors related to the culture medium were studied. Effects of medium composition on biomass growth and furanocoumarin content were analysed and optimal conditions were determined for phosphate (300 mg l⁻¹ of NaH₂PO₄), nitrate (2527 mg l⁻¹ of KNO₃), carbon source (10 g l⁻¹ of sucrose) and phytohormones (2,4-dichlorophenoxyacetic acid (2,4-D) 50 μM and benzylaminopurine (BAP) 50 μM). Ruta shoot growth and furanocoumarin production were compared for optimized and standard culture conditions. Specific medium gave better results in terms of growth (t_D equal to 6.9 days against 8.6 for standard conditions) but no significant differences appeared concerning metabolite concentrations. However, the present study opens the way to scale-up studies with bioreactor cultivation systems. This revised version was published online in June 2006 with corrections to the Cover Date.