Effect of carbon source on enzymes involved in glycerol metabolism inNeurospora crassa

Specific activities of eight enzymes involved in glycerol metabolism were determined in crude extracts of three strains ofNeurospora crassa after growth on six different carbon sources. One of the strains was wild type, which grew poorly on glycerol as sole carbon source; the other two were mutant strains which were efficient glycerol utilizers. A possible basis for this greater effeciency of glycerol utilization was catabolite repression of glyceraldehyde kinase by glycerol in wild type, and two-fold higher glycerate kinase activity in the mutant strains after growth on glycerol, thus apparently allowing two routes for glyceraldehyde to enter the glycolytic pathway in the mutant strains but only one in wild type. The preferential entry of glyceraldehyde to the glycolytic pathway through glycerate was suggested by the lack of glyceraldehyde kinase in all three strains after growth on one or more of the carbon sources and the generally higher levels of aldehyde dehydrogenase and of glycerate kinase than of glyceraldehyde kinase.     

Drug Effect Unveils Inter-head Cooperativity and Strain-dependent ADP Release in Fast Skeletal Actomyosin

Amrinone is a bipyridine compound with characteristic effects on the force-velocity relationship of fast skeletal muscle, including a reduction in the maximum shortening velocity and increased maximum isometric force. Here we performed experiments to elucidate the molecular mechanisms for these effects, with the additional aim to gain insight into the molecular mechanisms underlying the force-velocity relationship. In vitro motility assays established that amrinone reduces the sliding velocity of heavy meromyosin-propelled actin filaments by 30% at different ionic strengths of the assay solution. Stopped-flow studies of myofibrils, heavy meromyosin and myosin subfragment 1, showed that the effects on sliding speed were not because of a reduced rate of ATP-induced actomyosin dissociation because the rate of this process was increased by amrinone. Moreover, optical tweezers studies could not detect any amrinone-induced changes in the working stroke length. In contrast, the ADP affinity of acto-heavy meromyosin was increased about 2-fold by 1 mm amrinone. Similar effects were not observed for acto-subfragment 1. Together with the other findings, this suggests that the amrinone-induced reduction in sliding velocity is attributed to inhibition of a strain-dependent ADP release step. Modeling results show that such an effect may account for the amrinone-induced changes of the force-velocity relationship. The data emphasize the importance of the rate of a strain-dependent ADP release step in influencing the maximum sliding velocity in fast skeletal muscle. The data also lead us to discuss the possible importance of cooperative interactions between the two myosin heads in muscle contraction.Muscle contraction, as well as several other aspects of cell motility, results from cyclic interactions between myosin II motors and actin filaments. These force-generating interactions are driven by the hydrolysis of ATP at the myosin active site as outlined in Scheme 1 (1–3). In the absence of actin, the P_i and ADP release steps (k₄ and k₅) are rate-limiting for the entire cycle at high (>12 °C) and low temperatures, respectively (4–6). In the presence of actin, the rate of P_i release increases significantly, and the overall cycle is accelerated more than 2 orders of magnitude. The sliding velocity of myosin-propelled motors is generally believed to be rate-limited by actomyosin dissociation (rate constant k′₅, k′₆, or k′₂ in Scheme 1) (7). Alternatively, some studies (8, 9) have suggested that the sliding velocity is determined by the fraction of myosin heads in the weak-binding states, AM⁴ ATP and AM ADP P_i. However, it is worth emphasizing that K_T is very low under physiological conditions (1, 3) with low population of these states. For the same reason, the rate of dissociation of the AM complex is governed by K′₁ and k′₂.Open in a separate windowSCHEME 1.Simplified kinetics scheme for MgATP turnover by myosin (lower row) and actomyosin (upper row). Inorganic phosphate is denoted by P_i; MgATP is denoted by ATP, and MgADP is denoted by ADP; myosin is denoted by M. The states AM*ADP and AM ADP correspond to myosin heads with their nucleotide binding pocket in a partially closed and open conformation, respectively (7, 52). Rate constants are indicated by lowercase letters (rightward transitions, k₂ − k₅ and k′₂ − k′₅, or leftward transitions, k₋₂ − k₋₅ and k′₋₂ − k′₋₅) and equilibrium constants by uppercase letters (K₁, K′₁, K_T, K₃, K′₃, K₆, k′₆, and K_DP). The equilibrium constants are association constants except for simple bimolecular reactions where they are defined as k_i/k_−i.For the study of contractile mechanisms in both muscle and other types of cells, drugs may be useful as pharmacological tools affecting different transitions or states in the force-generating cycle. Whereas the use of drugs as tools may be less specific than site-directed mutagenesis, it also has advantages. The motor protein function may be studied in vivo, with maintained ordering of the protein components, e.g. as in the muscle sarcomere, allowing more insight into the relationship between specific molecular events and contractile properties of muscle. A drug that has been used quite extensively in this context is butanedione monoxime. The usefulness of this drug is based on firm characterization of its effect on actomyosin function on the molecular level (3, 10–13). More recently other drugs, like N-benzyl-p-toluene sulfonamide (14, 15) and blebbistatin (16), have been found to affect myosin function, and their effects at the molecular level have also been elucidated in some detail (14, 15, 17, 18). Both these drugs appear to affect the actomyosin interaction in a similar way as butanedione monoxime by inhibiting a step before (or very early in) the myosin power stroke, leading to the inhibition of actomyosin cross-bridge formation and force production.In contrast to the reduced isometric force, caused by the above mentioned drugs, the bipyridine compound amrinone (Fig. 1A) has been found to increase the isometric force production of fast intact skeletal muscles of the frog (19, 20) and mouse (21) and also of fast (but much less slow) skinned muscle fibers of the rat (22). In all the fast myosin preparations, the effect of about 1 mm amrinone on isometric force was associated with characteristic changes of the force-velocity relationship (Fig. 1B), including a reduced maximum velocity of shortening (19–22) and a reduced curvature of the force-velocity relationship (19–22). The latter effect was accompanied (20, 21) by a less pronounced deviation of the force-velocity relationship from the hyperbolic shape (23) at high loads. There have been different interpretations of the drug effects. It has been proposed (20–22) that amrinone might competitively inhibit the MgATP binding by myosin. However, more recently, results from in vitro motility assay experiments (24) challenged this idea. These results showed that amrinone reduces the sliding velocity (V_max) at saturating MgATP concentrations but not at MgATP concentrations close to, or below, the K_m value for the hyperbolic relationship between MgATP concentration and sliding velocity. Such a combination of effects is consistent with a reduced MgADP release rate (24) but not with competitive inhibition of substrate binding. However, effects of amrinone on the MgADP release rate have not been directly demonstrated. Additionally, in view of the uncertainty about what step actually determines the sliding velocity at saturating [MgATP] (see above and Refs. 7–9), it is of interest to consider other possible drug effects that could account for the data of Klinth et al. (24). These include the following: 1) an increased drag force, e.g. because of enhancement of weak actomyosin interactions; 2) a reduced step length; and 3) effects of the drug on the rate of MgATP-induced dissociation of actomyosin.Open in a separate windowFIGURE 1.A, structure of amrinone. B, experimental force-velocity data obtained in the presence (filled symbols) and absence (open symbols) of 1.1 mm amrinone. The data, from intact single frog muscle fibers, were obtained at 2 °C and fitted by Hill''s (42) hyperbola (lines) for data truncated at 80% of the maximum isometric force. Filled line, equation fitted to control data, a/P₀* = 0.185; P₀*/P₀ = 1.196. Dashed line, amrinone, a/P₀* = 0.347; P₀*/P₀ = 1.009. Force-velocity data were obtained in collaboration with Professor K. A. P. Edman. Same data as in Fig. 8 of Ref. 20. Note a decrease in maximum sliding velocity and curvature of the force-velocity relationship at low force, in response to amrinone. Also note that amrinone caused increased isometric force and a reduced deviation of the force-velocity relationship from the Hill''s hyperbola at high force. All changes of the force-velocity relationship were statistically significant (20), and similar changes were later also observed in intact mouse muscle and skinned rat muscle fibers. Data in Fig. 1 are published by agreement with Professor K. A. P. Edman.To differentiate between these hypotheses for the amrinone effects, and to gain more general insight into fundamental aspects of muscle function (e.g. mechanisms underlying the force-velocity relationship), we here study the molecular effects of amrinone on fast skeletal muscle myosin preparations in the presence and absence of actin.In vitro motility assay studies at different ionic strengths suggest that drag forces, caused by increased fraction of myosin heads in weak binding states, are not important for the effect of amrinone on sliding velocity. Likewise, optical tweezers studies showed no effect of the drug on the myosin step length. Finally, ideas that amrinone should reduce sliding velocity by reduced rate of MgATP-induced dissociation could be discarded because the drug actually increased the rate of this process. Instead, we found an amrinone-induced increase in the MgADP affinity of heavy meromyosin (HMM) in the presence of actin. Interestingly, similar effects of amrinone were not observed using myosin S1. As discussed below, this result and other results point to an amrinone-induced reduction in the rate of a strain-dependent MgADP release step. Simulations, using a model modified from that of Edman et al. (25), support this proposed mechanism of action. The results are discussed in relation to fundamental mechanisms underlying the force-velocity relationship of fast skeletal muscle, including which step determines shortening velocity and the possible importance of inter-head cooperativity.     

Responses to iron deficiency in Arabidopsis thaliana: The Turbo iron reductase does not depend on the formation of root hairs and transfer cells

Arabidopsis thaliana (L.) Heynh. Columbia wild type and a root hair-less mutant RM57 were grown on iron-containing and iron-deficient nutrient solutions. In both genotypes, ferric chelate reductase (FCR) of intact roots was induced upon iron deficiency and followed a Michaelis-Menten kinetic with a K _m of 45 and 54 M Fe^III-EDTA and a V _max of 42 and 33 nmol Fe²⁺·(g FW)^–1·min^–1 for the wild type and the mutant, respectively. The pH optimum for the reaction was around pH 5.5. The approximately four fold stimulation of FCR activity was independent of formation of root hairs and/or transfer cells induced by iron deficiency. Iron-deficiency-induced chlorosis and the development of a rigid root habit disappeared when ferric chelate was applied to the leaves, while FCR activity remained unchanged. The time course of the responses to iron deficiency showed that morphological and physiological responses were controlled separately.Abbreviations FCR ferric chelate reductase - FW fresh weight Thanks are due to Klaas Sjollema (Department of Electronmicroscopy, University of Groningen, The Netherlands) for help with the electron microscopy sample preparation and especially to Dr. Uwe Santore (Heinrich-Heine-University for electron microscopy. This work was supported by the SCIENCE programm of the European community; P.R.M.) and a Personal Research Grant by the Ministerium für Wissenschaft und Forschung of Nordrhein-Westfalen (P.R.M.) and last, not least by the productive discussions in ECOTRANS B.V.     

The phosphorus‐rich signature of fire in the soil–plant system: a global meta‐analysis

The biogeochemical and stoichiometric signature of vegetation fire may influence post‐fire ecosystem characteristics and the evolution of plant ‘fire traits’. Phosphorus (P), a potentially limiting nutrient in many fire‐prone environments, might be particularly important in this context; however, the effects of fire on P cycling often vary widely. We conducted a global‐scale meta‐analysis using data from 174 soil studies and 39 litter studies, and found that fire led to significantly higher concentrations of soil mineral P as well as significantly lower soil and litter carbon:P and nitrogen:P ratios. These results demonstrate that fire has a P‐rich signature in the soil–plant system that varies with vegetation type. Further, they suggest that burning can ease P limitation and decouple the biogeochemical cycling of P, carbon and nitrogen. These effects resemble a transient reversion to an earlier stage of ecosystem development, and likely underpin at least some of fire's impacts on ecosystems and organisms.     

An analysis of spontaneous behavior following acute MDMA treatment in male and female rats

3,4-methylenedioxymethamphetamine (MDMA, ecstasy) is a widely abused drug that impairs behavioral, emotional and cognitive functions in humans and animals. The aim of this study was to evaluate MDMA effects on the spontaneous behavioral repertoire of rats with a focus on the gender differences. MDMA was given subcutaneously in a single dose of 2.5, 5 and 10 mg/kg and the spontaneous behavior of male and female rats was studied using the open field test. Behavioral patterns (locomotion, rearing, floor-sniffing, air-sniffing, grooming, immobility and stereotypy) were registered in two sessions - 30 and 60 min following MDMA administration; each session lasting 5 min. We found that MDMA totally disrupted the structure and timing of spontaneous behavioral patterns in both genders; no evident differences were measured between either of the sessions. MDMA irrespective of the dosage produced hyperlocomotion, excessive floor-sniffing and almost absolute suppression of grooming and immobility. A biphasic effect of MDMA was found in rearing. Gender differences were present namely in rearing and sniffing stereotypy. This study also confirms that behavioral experiments should focus on more behavioral elements than only on e.g. locomotion and that the observer-based approach still gives the most reliable results.     

THE CELLULAR IMMUNE RESPONSE OF DAPHNIA MAGNA UNDER HOST–PARASITE GENETIC VARIATION AND VARIATION IN INITIAL DOSE

In invertebrate–parasite systems, the likelihood of infection following parasite exposure is often dependent on the specific combination of host and parasite genotypes (termed genetic specificity). Genetic specificity can maintain diversity in host and parasite populations and is a major component of the Red Queen hypothesis. However, invertebrate immune systems are thought to only distinguish between broad classes of parasite. Using a natural host–parasite system with a well‐established pattern of genetic specificity, the crustacean Daphnia magna and its bacterial parasite Pasteuria ramosa, we found that only hosts from susceptible host–parasite genetic combinations mounted a cellular response following exposure to the parasite. These data are compatible with the hypothesis that genetic specificity is attributable to barrier defenses at the site of infection (the gut), and that the systemic immune response is general, reporting the number of parasite spores entering the hemocoel. Further supporting this, we found that larger cellular responses occurred at higher initial parasite doses. By studying the natural infection route, where parasites must pass barrier defenses before interacting with systemic immune responses, these data shed light on which components of invertebrate defense underlie genetic specificity.     

Intravascular plug formation induced by poly-APS is the principal mechanism of the toxin's lethality in rats/rat tissues

Toxic water soluble polymeric 3-alkylpyridinium salts isolated from the sponge Raniera sarai strongly inhibited AChE in vitro. In vivo, experimental animals died due to plugs formed in microcirculation. The mechanism of this plug formation is unknown. In vitro, the toxin did not affect the coagulation rate, but the rate of platelet aggregation was accelerated in a dose-dependent manner. The hemolytic activity of poly-APS was diminished by the addition of serum proteins in a dose-dependent manner. These results support the conclusion that non-specific binding to proteins is the underlying mechanism of the lethality of poly APS.