Author Correction: Crowther et al. reply.

In this Brief Communications Arising Reply, the affiliation for author P. H. Templer was incorrectly listed as 'Department of Ecology & Evolutionary Biology, University of California Irvine, Irvine, California 92697, USA' instead of 'Department of Biology, Boston University, Boston, Massachusetts 02215, USA'. This has been corrected online.

Quantifying global soil carbon losses in response to warming.

The majority of the Earth's terrestrial carbon is stored in the soil. If anthropogenic warming stimulates the loss of this carbon to the atmosphere, it could drive further planetary warming. Despite evidence that warming enhances carbon fluxes to and from the soil, the net global balance between these responses remains uncertain. Here we present a comprehensive analysis of warming-induced changes in soil carbon stocks by assembling data from 49 field experiments located across North America, Europe and Asia. We find that the effects of warming are contingent on the size of the initial soil carbon stock, with considerable losses occurring in high-latitude areas. By extrapolating this empirical relationship to the global scale, we provide estimates of soil carbon sensitivity to warming that may help to constrain Earth system model projections. Our empirical relationship suggests that global soil carbon stocks in the upper soil horizons will fall by 30 ± 30 petagrams of carbon to 203 ± 161 petagrams of carbon under one degree of warming, depending on the rate at which the effects of warming are realized. Under the conservative assumption that the response of soil carbon to warming occurs within a year, a business-as-usual climate scenario would drive the loss of 55 ± 50 petagrams of carbon from the upper soil horizons by 2050. This value is around 12-17 per cent of the expected anthropogenic emissions over this period. Despite the considerable uncertainty in our estimates, the direction of the global soil carbon response is consistent across all scenarios. This provides strong empirical support for the idea that rising temperatures will stimulate the net loss of soil carbon to the atmosphere, driving a positive land carbon-climate feedback that could accelerate climate change.

Plastic detection comb better than visual screening for diagnosis of head louse infestation.

Finding lice can be difficult in head louse infestation. We compared a new louse detection comb with visual inspection. All children in two rural Turkish schools were screened by the two methods. Those with lice were offered treatment and the results monitored by detection combing. Children with nits only were re-screened to identify latent infestations. Using visual inspection we found 214/461 children (46%) with nits but only 30 (6.5%) with live lice. In contrast detection combing found 96 (21%) with live lice, of whom 20 had no nits. Detection combing was 3.84 times more effective than visual inspection for finding live lice. Only 10/138 (7.2%) children with nits and no lice were found to have active infestation by day 16. We found that the detection comb is significantly (P<0.001) more effective than visual screening for diagnosis; that nits are not a good indicator of active infestation; and that treatment with 1% permethrin was 89.6% effective.

Efficacy of imidacloprid, imidacloprid/permethrin and phoxim for flea control in the Mustelidae (ferrets, mink).

Farmed mink (Mustela vison), a close relative of the domestic ferret (Mustela putorius furo), naturally infested with the squirrel flea (Ceratophyllus sciurorum) were included in a study to investigate three compounds for flea control. The test products were imidacloprid in a 10% (w/v) solution, an imidacloprid 10% (w/v)/permethrin 50% (w/v) solution, and phoxim; all three are well-known compounds for the control of different ectoparasites in a wide range of animals. Two groups of mink received 0.1 ml per animal of the imidacloprid or the imidacloprid/permethrin combination at days 0 and 28, respectively. Two groups of mink were sprayed with 25 ml of a 0.1% phoxim solution at day 0 and either 1x25 ml or 2x25 ml, respectively, of a 0.05% phoxim solution at day 28. One group of mink served as an untreated control. At assessment on day 56 the efficacy was 91.9% in the imidacloprid group, 89.3% in the imidacloprid/permethrin group, 92.2% in the phoxim 1x25-ml group and 99.3% in the phoxim 2x25 ml group, respectively. In the untreated control group an average of 757 fleas per mink nesting material was recorded.

Infestation of Werneckiella equi on Icelandic horses, characteristics of predilection sites and lice dermatitis.

Lice infestations on horses caused by the lice Werneckiella (Damalinia) equi and Haematopinus equi are observed worldwide. In this study, the distribution and clinical manifestations of lice on Icelandic horses were examined. Thirty-eight out of 93 animals (40.86%) were identified as infested with W. equi. Sixty-eight animals (73.12%) presented dermatological lesions associated with lice infestation, while only 32 of these animals presented lice. Six animals had no clinical signs although of being lice-positive, and 19 animals (20.43%) showed neither lice nor clinical manifestations. Lice burdens varied from animal to animal, and clinical manifestations occurred at all levels of infestation. Focal alopecia was the main clinical sign (83.78%) on lice-positive horses, while scaling and crusts occurred in 10.81% and 9.68% of the cases, respectively. Clinical signs present in the head and the neck/mane area were found to be an indication of lice infestation in horses.

Dermatological and parasitological evaluation of infestations with chewing lice (Werneckiella equi) on horses and treatment using imidacloprid.

Lice infestations in horses caused by the chewing louse Werneckiella (Damalinia) equi are observed worldwide. In the present study, the efficacy of 10% imidacloprid was tested on horses naturally infested with lice. Two groups of animals received a double application of 4 ml and 8 ml Advantage 10% spot-on on day 0 and 28 either. Horses, presenting dermatological signs with negative lice counts, were also included in this investigation. 40.86% of the horses presented positive lice counts and 84.21% of these animals showed clinical dermatologic signs. 65.45% of the lice-negative horses also showed clinical manifestations. Two days after treatment, lice counts dropped in both the treatment groups and on day 56, all animals were free of alive lice, and dermatological lesions decreased significantly (P<0.001) in both the lice-positive and the negative animals. No correlation (P>0.050) between lice burden and clinical signs was detected.

Natural infestation of the chewing lice (Werneckiella equi) on horses and treatment with imidacloprid and phoxim.

Infestation with the chewing louse (Werneckiella (Damalinia) equi) can be found on horses world-wide. Louse infestations, including clinical signs of louse-derived dermatitis, are known from Icelandic horses. A clinical field investigation was conducted in Iceland using horses with natural louse infestations to evaluate the efficacy of imidacloprid in a 10% solution in comparison with phoxim in a 0.05% solution. A total of 27 horses received a single imidacloprid treatment using 16 ml of the 10% solution along the mane and on the dorso-lateral trunk. A further 43 horses were treated twice, 14 days apart, with phoxim, using 2 x 50 ml solution applied along the mane and the dorso-lateral trunk. At the final evaluation on day 28, complete control of the lice was obtained for the imidacloprid treated horses and only a single moribund louse was found on two horses treated with phoxim.

Host location, survival and fecundity of the Oriental rat flea Xenopsylla cheopis (Siphonaptera: Pulicidae) in relation to black rat Rattus rattus (Rodentia: Muridae) host age and sex.

Host choice and fecundity are two factors that may contribute to the variation in flea counts observed when assessing the potential risk of flea-borne transmission of pathogens from rodents to humans. Using the black rat, Rattus rattus Linnaeus, as host the effects of age and sex on host choice and fecundity of the Oriental rat flea, Xenopsylla cheopis Rothschild, were examined experimentally at 25 degrees C and 80% rh. During the first two days of emergence from cocoons, female fleas dominated the sex ratio by 4:1 but from the third day onwards this switched to a male-dominated sex ratio of 4:1. The sex of the flea did not influence their host-seeking behaviour. Newly emerged fleas of both sexes were not influenced by the rat's presence and at seven days old both sexes demonstrated similar levels of attraction toward the rat host. The sex of the rat did not affect flea host-seeking behaviour. There was a 50-70% decline in the initial number of adult fleas during the first week after their release onto a rat host, and this decline was greatest on juvenile rats. Flea fecundity was also significantly lower on juvenile rat hosts but no differences due to the sex of the rat were observed. This experimental study supports the hypothesis that differences in flea count due to host sex, reported in field surveys, result from sexual differences in host behaviour and not from discriminatory host-seeking behaviour by X. cheopis. Differences in flea count due to host age may be affected by differences in X. cheopis fecundity, which may itself be mediated by host behaviour such as grooming.

Palatability and toxicity of fipronil as a systemic insecticide in a bromadiolone rodenticide bait for rat and flea control.

Vector control in plague-infested areas requires a simultaneous killing of rodents and their fleas. We investigated the efficacy of a combination of a systemic insecticide, fipronil, in a rodenticide bait formulation under laboratory conditions. Four different concentrations of fipronil (0.05%, 0.005%, 0.0005% with acetone as a solvent, and 0.05% with propylene glycol as a solvent) and two controls (solvents only) were combined with the rodenticide bait (crushed organically grown wheat with 0.005% bromadiolone). Each concentration was offered together with an untreated non-poisonous challenge bait to 10 singly caged Rattus rattus L., each with 100 rat fleas Xenopsylla cheopis Rothschild (Siphonaptera: Pulicidae) in the nest. Treated bait consumption was relatively low and an unsatisfactory rat mortality of around 50% only was obtained in all tests. The palatability of the bait, however, was not affected by the fipronil concentration. Even at the lowest fipronil concentration, average flea mortality was still above 95%, and doses of more than I mg fipronil per kg rat body weight gave a nearly complete kill of fleas. Fipronil can be highly effective as a systemic insecticide to for flea control, provided that a more attractive bait base for roof rats is used.

A circadian rhythm of locomotor activity in newly emerged Ceratophyllus sciurorum.

Circadian rhythm in newly emerged individuals of the Red Squirrel (Scuirus vulgaris) flea C.s.sciurorum was studied in a constant environment, using an insect activity monitor. Trials were run over 7 days using two start times (08.00 and 17.00 hours). The results show that, regardless of start time, the fleas display a 24 h activity rhythm. The presence of a rhythm under constant conditions gives a strong indication that C.s.sciurorum has a self-sustaining clock which is started by disturbance and is most likely to be linked to host activity patterns.

Coordination dynamics of biological zinc "clusters" in metallothioneins and in the DNA-binding domain of the transcription factor Gal4.

The almost universal appreciation for the importance of zinc in metabolism has been offset by the considerable uncertainty regarding the proteins that store and distribute cellular zinc. We propose that some zinc proteins with so-called zinc cluster motifs have a central role in zinc distribution, since they exhibit the rather exquisite properties of binding zinc tightly while remaining remarkably reactive as zinc donors. We have used zinc isotope exchange both to probe the coordination dynamics of zinc clusters in metallothionein, the small protein that has the highest known zinc content, and to investigate the potential function of zinc clusters in cellular zinc distribution. When mixed and incubated, metallothionein isoproteins-1 and -2 rapidly exchange zinc, as demonstrated by fast chromatographic separation and radiometric analysis. Exchange kinetics exhibit two distinct phases (k(fast) approximately 5000 min(-1) x M(-1); k(slow) approximately 200 min(-1) x M(-1), pH 8.6, 25 degrees C) that are thought to reflect exchange between the three-zinc clusters and between the four-zinc clusters, respectively. Moreover, we have observed and examined zinc exchange between metallothionein-2 and the Gal4 protein (k approximately 800 min(-1) x M(-1), pH 8.0, 25 degrees C), which is a prototype of transcription factors with a two-zinc cluster. This reaction constitutes the first experimental example of intermolecular zinc exchange between heterologous proteins. Such kinetic reactivity distinguishes zinc in biological clusters from zinc in the coordination environment of zinc enzymes, where the metal does not exchange over several days with free zinc in solution. The molecular organization of these clusters allows zinc exchange to proceed through a ligand exchange mechanism, involving molecular contact between the reactants.

D-Phe complexes of zinc and cobalt carboxypeptidase A.

The binding of D-phenylalanine, D-Phe, to both zinc and cobalt carboxypeptidase A, ZnCPD and CoCPD, has been investigated by a combination of kinetic and spectroscopic techniques. Kinetic studies of the ZnCPD catalyzed hydrolysis of dansyl-Gly-Ala-L-Phe indicate that D-Phe inhibition occurs through a two-site sequential competitive inhibition mode with Ki values of 45 microM and 11.6 mM at pH 8.4, 1 M NaCl, 25 degrees C. Spectral titration of CoCPD under the same conditions indicates a very strong binding mode of D-Phe (KD < 100 microM) that only slightly perturbs the visible cobalt electronic transitions. However, the conversion of CoCPD.D-Phe into a CoCPD.D-Phe2 (KD, 1.13 mM) is accompanied by a very strong spectral perturbation resulting in a complex that is characterized by Amax values of 506 nm (epsilon = 27 M-1 cm-1) and 605 nm (epsilon = 17 M-1 cm-1) and a shoulder at 530 nm (epsilon = 23 M-1 cm-1). The spectral properties of this ternary complex differ markedly from that of the CoCPD.L-Phe.N3-ternary complex. X-ray absorption fine structure, XAFS, studies indicate that these differences are likely due to a more regular tetrahedral coordination sphere for the ternary azide complexes compared to an octahedral coordination geometry for the Zn and CoCPD.D-Phe2 complexes.

X-ray absorption fine structure study of the active site of zinc and cobalt carboxypeptidase A in their solution and crystalline forms.

A comparative study on the metal environment of Zn(II)-carboxypeptidase A (ZnCPD) and Co(II)-carboxypeptidase A (CoCPD) in their solution and crystalline forms using the X-ray absorption fine structure (XAFS) technique has been conducted. The first coordination sphere of Zn for ZnCPD in its solution state is found to consist of two distributions of atoms, with four atoms (N or O) located at an average distance of 2.03 +/- 0.01 A and one atom (N or O) located at 2.57 +/- 0.04 A. The four-atom distribution remains the same for ZnCPD in its crystalline state, but the fifth atom is found at 2.36 +/- 0.04 A. Examination of the higher coordination shell, between 2.7 and 4.2 A, reveals the presence of two imidazoles. Combined with X-ray crystallographic results, a structural model is proposed. The four atoms at an average distance of 2.03 A are assigned to the two delta 1 nitrogens of His-69 and His-196, one epsilon 1 oxygen of Glu-72, and the oxygen of a coordinated water molecule. The atom at 2.57 A for ZnCPD in solution is assigned to the epsilon 2 oxygen of Glu-72. The results for CoCPD in solution are similar with the four atoms at an average distance of 2.08 +/- 0.01 A and one atom at 2.50 +/- 0.04 A, which moves to 2.34 +/- 0.04 A in the crystalline enzyme. The intensity of the 3d "pip" peak for CoCPD is consistent with a distorted tetragonal metal geometry for the solution form of the enzyme which is converted to a more pentacoordinated metal site for the crystalline enzyme. The first shell distribution of crystalline CoCPD is quite disordered, which may be largely due to the disorder of His-69 and His-196 as indicated by higher shell analysis. Thus, the XAFS studies show that the metal coordination spheres in the zinc and cobalt enzymes are quite similar in the solution state but differ from their crystalline counterparts. The XAFS studies provide the necessary background for measurement of substrate- and inhibitor-promoted structural changes in the metal coordination sphere of the zinc and other metal-substituted carboxypeptidases in the solution state.

Characterization of an inhibitory metal binding site in carboxypeptidase A.

The specificity of metal ion inhibition of bovine carboxypeptidase A ([(CPD)Zn]) catalysis is examined under stopped-flow conditions with use of the fluorescent peptide substrate Dns-Gly-Ala-Phe. The enzyme is inhibited competitively by Zn(II), Pb(II), and Cd(II) with apparent KI values of 2.4 x 10(-5), 4.8 x 10(-5), and 1.1 x 10(-2) M in 0.5 M NaCl at pH 7.5 and 25 degrees C. The kcat/Km value, 7.3 x 10(6) M-1 s-1, is affected less than 10% at 1 x 10(-4) M Mn(II) or Cu(II) and at 1 x 10(-2) M Co(II), Ni(II), Hg(II), or Pt(IV). Zn(II) and Pb(II) are mutually exclusive inhibitors. Previous studies of the pH dependence of Zn(II) inhibition [Larsen, K. S., & Auld, D. S. (1989) Biochemistry 28, 9620] indicated that [(CPD)Zn] is selectively inhibited by a zinc monohydroxide complex, ZnOH+, and that ionization of a ligand, LH, in the enzyme's inhibitory site (pKLH 5.8) is obligatory for its binding. The present study allows further definition of this inhibitory zinc site. The ionizable ligand (LH) is assigned to Glu-270, since specific chemical modification of this residue decreases the binding affinity of [(CPD)Zn] for Zn(II) and Pb(II) by more than 60- and 200-fold, respectively. A bridging interaction between the Glu-270-coordinated metal hydroxide and the catalytic metal ion is implicated from the ability of Zn(II) and Pb(II) to induce a perturbation in the electronic absorption spectrum of cobalt carboxypeptidase A ([(CPD)Co]).(ABSTRACT TRUNCATED AT 250 WORDS)

Catalytic and conformational changes induced by limited subtilisin cleavage of bovine carboxypeptidase A.

Limited proteolysis of carboxypeptidase A from bovine pancreas with subtilisin Carlsberg generates a stable intermediate, carboxypeptidase S, whose esterase and peptidase activities are increased and decreased, respectively, under standard assay conditions. Carboxypeptidase S was isolated by affinity chromatography. Sequence analysis shows that it is cleaved solely at the Ala154-Gly155 bond. Its enzymatic properties were determined under stopped-flow conditions with Dns-Gly-Ala-Phe and its ester analogue Dns-Gly-Ala-OPhe. For both substrates, the Km values are increased 30-40-fold. The kcat value for peptide hydrolysis is virtually unaffected whereas that for ester hydrolysis is increased 10-fold. The magnitude of the Km effect is equivalent to a loss of 9 kJ/mol of binding energy and likely reflects a disruption of the network of hydrogen bonds that links Tyr-248 and Arg-145 to the backbone carbonyls of Ala-154 and Gly-155. The difference in kcat effects for the two substrate classes is related to differences in the chemical nature of the rate-determining step. Product release is rate determining for catalytic hydrolysis of ester substrates, and hence, the increase in kcat indicates that dissociation of products is facilitated as a result of the Ala154-Gly155 bond scission. The changes in enzymatic activity accompanying limited proteolysis are due to conformational alterations in the vicinity of the active center of the molecule. The affinity of a monoclonal antibody, mAb 100, directed toward the antigenic determinant located between residues 209 and 218 in carboxypeptidase A is diminished considerably for carboxypeptidase S.(ABSTRACT TRUNCATED AT 250 WORDS)