Impact on short-lived climate forcers (SLCFs) from a realistic land-use change scenario via changes in biogenic emissions.

More than one quarter of natural forests have been cleared by humans to make way for other land-uses, with changes to forest cover projected to continue. The climate impact of land-use change (LUC) is dependent upon the relative strength of several biogeophysical and biogeochemical effects. In addition to affecting the surface albedo and exchanging carbon dioxide (CO2) and moisture with the atmosphere, vegetation emits biogenic volatile organic compounds (BVOCs), altering the formation of short-lived climate forcers (SLCFs) including aerosol, ozone (O3) and methane (CH4). Once emitted, BVOCs are rapidly oxidised by O3, and the hydroxyl (OH) and nitrate (NO3) radicals. These oxidation reactions yield secondary organic products which are implicated in the formation and growth of aerosol particles and are estimated to have a negative radiative effect on the climate (i.e. a cooling). These reactions also deplete OH, increasing the atmospheric lifetime of CH4, and directly affect concentrations of O3; the latter two being greenhouse gases which impose a positive radiative effect (i.e. a warming) on the climate. Our previous work assessing idealised deforestation scenarios found a positive radiative effect due to changes in SLCFs; however, since the radiative effects associated with changes to SLCFs result from a combination of non-linear processes it may not be appropriate to scale radiative effects from complete deforestation scenarios according to the deforestation extent. Here we combine a land-surface model, a chemical transport model, a global aerosol model, and a radiative transfer model to assess the net radiative effect of changes in SLCFs due to historical LUC between the years 1850 and 2000.

Impact on short-lived climate forcers increases projected warming due to deforestation.

The climate impact of deforestation depends on the relative strength of several biogeochemical and biogeophysical effects. In addition to affecting the exchange of carbon dioxide (CO2) and moisture with the atmosphere and surface albedo, vegetation emits biogenic volatile organic compounds (BVOCs) that alter the formation of short-lived climate forcers (SLCFs), which include aerosol, ozone and methane. Here we show that a scenario of complete global deforestation results in a net positive radiative forcing (RF; 0.12 W m-2) from SLCFs, with the negative RF from decreases in ozone and methane concentrations partially offsetting the positive aerosol RF. Combining RFs due to CO2, surface albedo and SLCFs suggests that global deforestation could cause 0.8 K warming after 100 years, with SLCFs contributing 8% of the effect. However, deforestation as projected by the RCP8.5 scenario leads to zero net RF from SLCF, primarily due to nonlinearities in the aerosol indirect effect.

Solution structure of the cardiostimulant polypeptide anthopleurin-B and comparison with anthopleurin-A.

BACKGROUND: The polypeptide anthopleurin-B (AP-B) is one of a number of related toxins produced by sea anemones. AP-B delays inactivation of the voltage-gated sodium channel of excitable tissue. In the mammalian heart, this effect is manifest as an increase in the force of contraction. As a result, there is interest in exploiting the anthopleurins as lead compounds in the design of novel cardiac stimulants. Essential to this endeavour is a high-resolution solution structure of the molecule describing the positions of functionally important side chains. RESULTS: AP-B exists in multiple conformations in solution as a result of cis-trans isomerization about the Gly40-Pro41 peptide bond. The solution structure of the major conformer of AP-B has been determined by two-dimensional 1H NMR at pH 4.5 and 25 degrees C. The core structure is a four-stranded, antiparallel beta-sheet (residues 2-4, 20-23, 34-37 and 45-48) and includes several beta-turns (6-9, 25-28, 30-33). Three loops connect the beta-strands, the longest and least well defined being the first loop, extending from residues 8-17. These features are shared by other members of this family of sea anemone toxins. The locations of a number of side chains which are important for the cardiac stimulatory activity of AP-B are well defined in the structures. CONCLUSIONS: We have described the solution structure of AP-B and compared it with that of AP-A, from which it differs by substitutions at seven amino acid positions. It shares an essentially identical fold with AP-A yet is about 10-fold more active. Comparison of the structures, particularly in the region of residues essential for activity, gives a clearer indication of the location and extent of the cardioactive pharmacophore in these polypeptides.

Limited proteolysis study of structure-function relationships in Sh I, a polypeptide neurotoxin from a sea anemone.

The structure-function relationships of the neurotoxic polypeptide Sh I, from the sea anemone Stichodactyla helianthus, have been studied using limited proteolysis with trypsin and endoproteinase Lys-C. Major products from each of the proteolytic digests were characterised using N-terminal peptide sequencing and amino-acid analysis or mass spectrometry. Of the six possible tryptic cleavage sites in Sh I, the bonds adjacent to Arg-13 and Lys-47 were found to be the most susceptible, complete cleavage occurring within minutes. Cleavages adjacent to Lys-32 and Lys-46 proceeded more slowly and cleavage adjacent to Arg-45 was the slowest. The sixth potential site, adjacent to Lys-4, was not cleaved at all. All derivatives were inactive as crustacean neurotoxins. Cleavage with endoproteinase Lys-C generated two major products. Derivatives cleaved adjacent to Lys-32 and either Lys-46 or Lys-47 were isolated. Both were inactive, indicating that either cleavage adjacent to Lys-32 or the removal of the C-terminal lysine residue(s) was sufficient to abolish activity. Lys-4 again was refractory to cleavage. The sequence of cleavage events correlated well with the static accessibility of the lysyl and arginyl side chains and to a lesser extent with the accessibility of the carbonyl oxygen of susceptible peptide bonds, as measured from the solution structure of Sh I determined by 1H-NMR. In the case of Lys-4, the lack of cleavage by trypsin and endoproteinase Lys-C may reflect a lack of flexibility in this region. The effects of the various cleavages on biological activity emphasise that the surface of the protein near the reverse turn encompassing Asp-6, Asp-7 and Glu-8 is essential for activity.

Helical structure and packing orientation of the S2 segment in the Shaker K+ channel.

Six transmembrane segments, S1-S6, cluster around the central pore-forming region in voltage-gated K+ channels. To investigate the structural characteristics of the S2 segment in the Shaker K+ channel, we replaced each residue in S2 singly with tryptophan (or with alanine for the native tryptophan). All but one of the 23 Trp mutants expressed voltage-dependent K+ currents in Xenopus oocytes. The effects of the mutations were classified as being of low or high impact on channel gating properties. The periodicity evident in the effects of these mutations supports an alpha-helical structure for the S2 segment. The high- and low-impact residues cluster onto opposite faces of a helical wheel projection of the S2 segment. The low-impact face is also tolerant of single mutations to asparagine. All results are consistent with the idea that the low-impact face projects toward membrane lipids and that changes in S2 packing occur upon channel opening. We conclude that the S2 segment is a transmembrane alpha helix and that the high-impact face packs against other transmembrane segments in the functional channel.

Toxins from the box-jellyfish Chironex fleckeri.

Two myotoxins (T1 and T2) with mol. wts of approximately 600,000 and 150,000, respectively, and a haemolysin (T3) with a mol. wt of approximately 70,000 were isolated from the crude nematocyst venom of C. fleckeri by the use of Sephadex G-200 chromatography. A neurotoxic fraction (T4) and a haemolytic fraction (T5) containing proteins with apparent mol. wts of approximately 150,000 and 70,000, respectively, were also isolated by Sephadex chromatography from crude extracts of tentacular material from which nematocysts had been removed. The three nematocyst toxins and the two toxic fractions from tentacle extracts were lethal to mice on i.v. injection. After SDS-PAGE the myotoxins T1 and T2 yielded similar major bands corresponding with mol. wts different from those yielded by T3 and the toxic tentacle fractions. T1 and T2 appeared to be comprised of aggregations of subunits with mol. wts of approximately 18,000. On HPLC, crude nematocyst venom and the nematocyst toxins T1 and T2 lost their myotoxic properties. The need for thorough removal of extraneous tentacular material from isolated nematocysts, the need for effective rupture of nematocysts, the need to counter the lability of the nematocyst venom and the need to use myotoxicity as a criterion of venom activity if the active components of the venom are to be purified and characterized are emphasized.

Apparent relationships between toxins elaborated by the cyanobacterium Trichodesmium erythraeum and those present in the flesh of the narrow-barred Spanish mackerel Scomberomorus commersoni.

The marine cyanobacterium Trichodesmium erythraeum contains toxic water-soluble material that produces signs in mice similar to those produced by water-soluble extracts of the flesh of a specimen of pelagic fish Scomberomorus commersoni from a batch that had been implicated in a poisoning resembling ciguatera. Extracts of water-soluble material from both the cyanobacterium and the fish contained toxins that were chromatographically indistinguishable. A peptide and an alkaloid were detected in partially purified extracts of the water-soluble material. In addition to this material toxic lipid-soluble material was present in some batches of T. erythraeum. Elution of this material with 9:1 chloroform:methanol using column chromatography produced material that was chromatographically indistinguishable from ciguatoxin-like material from S. commersoni and produced signs in mice similar to those produced by this material. Elution of the lipid-soluble material with 97:3 chloroform:methanol yielded a toxin resembling in its chromatographic and toxic properties a scaritoxin-like substance from S. commersoni. Other toxins with Rf values lying between that of the ciguatoxin-like material and that of the scaritoxin-like material were also detected in extracts of T. erythraeum. It is postulated that T. erythraeum is the progenitor of major toxins carried by some ciguateric fish and that water-soluble toxins released into the ambient sea water by T. erythraeum may constitute a health hazard for humans.

Multiple toxins in a specimen of the narrow-barred Spanish mackerel, Scomberomorus commersoni.

Ciguatoxin-like, scaritoxin-like and other unidentified lipid-soluble toxins were detected in a specimen of Scomberomorus commersoni captured in sub-tropical Queensland. The fish was from a batch that had been involved in human poisonings. The ciguatoxin-like substance made a greater contribution to the total toxicity than did the scaritoxin-like substance. Water-soluble toxins were also present. The most important of these in terms of contribution to total toxicity was unidentified toxic material present in fractions eluted from a silicic acid column with 100% methanol or methanol:water (1:1). After TLC this material yielded a spot positive for alkaloids. Maitotoxin was also detected among the water-soluble toxins. The lethal potency of the fish flesh approximated 27.3 MU/100 g of flesh with water-soluble toxins contributing to a greater extent than the lipid-soluble toxins. (A MU is defined as the minimum amount of toxic material expressed in g required to kill a 20 g mouse within 24 hr after i.p. injection.) The 15 kg fish studied contained a total of approximately 4095 MU. The presence of several water-soluble and lipid-soluble toxins in the fish has implications for the detection of such ciguateric fishes and for the diagnosis and treatment of poisonings stemming from ingestion of these fishes.

Variation in the toxins present in ciguateric narrow-barred Spanish mackerel, Scomberomorus commersoni.

Water-soluble and lipid-soluble toxins present in six specimens of Scomberomorus commersoni captured in subtropical Queensland were compared with those detected in a specimen studied earlier. All specimens were from batches that had been involved in human poisonings. All specimens contained significant amounts of potent water-soluble toxins, the most important of which in terms of contribution to the lethal potency of fish flesh was unidentified toxic material which tested positively for alkaloids. All specimens contained lipid-soluble toxins including ciguatoxin-like and scaritoxin-like material, the latter usually predominating. Amounts of water-soluble toxins with lethal potencies ranging from 14.9 MU to 115 MU/100 g of flesh and of lipid-soluble toxins with lethal potencies ranging from 8.8 MU to 39.9 MU/100 g of flesh were found. (A mouse unit, MU, is the minimum amount of toxic material expressed in g required to kill a 20 g mouse within 24 hr following i.p. injection.) The lethal potency of water-soluble toxins per g of fish exceeded that of lipid-soluble toxins per g of fish for five of the seven specimens of S. commersoni now investigated. Based on a lethal dose to humans of 2500 MU all fishes contained lethal amounts of toxic material. The relative amounts of water-soluble and lipid-soluble toxins present in the flesh of a specimen of S. commersoni were altered by different cooking procedures.

Removing the sampling restrictions from family-based tests of association for a quantitative-trait locus.

One strategy for localization of a quantitative-trait locus (QTL) is to test whether the distribution of a quantitative trait depends on the number of copies of a specific genetic-marker allele that an individual possesses. This approach tests for association between alleles at the marker and the QTL, and it assumes that association is a consequence of the marker being physically close to the QTL. However, problems can occur when data are not from a homogeneous population, since associations can arise irrespective of a genetic marker being in physical proximity to the QTL-that is, no information is gained regarding localization. Methods to address this problem have recently been proposed. These proposed methods use family data for indirect stratification of a population, thereby removing the effect of associations that are due to unknown population substructure. They are, however, restricted in terms of the number of children per family that can be used in the analysis. Here we introduce tests that can be used on family data with parent and child genotypes, with child genotypes only, or with a combination of these types of families, without size restrictions. Furthermore, equations that allow one to determine the sample size needed to achieve desired power are derived. By means of simulation, we demonstrate that the existing tests have an elevated false-positive rate when the size restrictions are not followed and that a good deal of information is lost as a result of adherence to the size restrictions. Finally, we introduce permutation procedures that are recommended for small samples but that can also be used for extensions of the tests to multiallelic markers and to the simultaneous use of more than one marker.

A new paradigm for drug discovery: integrating clinical, genetic, genomic and molecular phenotype data to identify drug targets.

Application of statistical genetics approaches to variations in mRNA transcript abundances in segregating populations can be used to identify genes and pathways associated with common human diseases. The combination of this genetic information with gene expression and clinical trait data can also be used to identify subtypes of a disease and the genetic loci specific to each subtype. Here we highlight results from some of our recent work in this area and further explore the many possibilities that exist in employing a more comprehensive genetics and functional genomics approach to the functional annotation of genomes, and in applying such methods to the validation of targets for complex traits in the drug discovery process.

A comparative study of sibship tests of linkage and/or association.

Population-based tests of association have used data from either case-control studies or studies based on trios (affected child and parents). Case-control studies are more prone to false-positive results caused by inappropriate controls, which can occur if, for example, there is population admixture or stratification. An advantage of family-based tests is that cases and controls are well matched, but parental data may not always be available, especially for late-onset diseases. Three recent family-based tests of association and linkage utilize unaffected siblings as surrogates for untyped parents. In this paper, we propose an extension of one of these tests. We describe and compare the four tests in the context of a complex disease for both biallelic and multiallelic markers, as well as for sibships of different sizes. We also examine the consequences of having some parental data in the sample.

Preparation and characterization of a biologically active spin-labeled sea anemone toxin.

A derivative of the polypeptide cardiostimulant anthopleurin-B(AP-B) labeled with the spin label 1-oxyl 2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl azide has been prepared and characterized. The product was found by mass spectrometry to be labeled at a single site, which amino acid sequencing showed to be the N-terminus. It also retained positive inotropic activity when assayed on isolated guinea pig atria. The spin-labeled (SL) product was found to exist in two distinct conformations by reversed-phase HPLC and in at least two conformations by electron spin resonance spectroscopy (ESR) over the pH range 2-9. The ESR data also show evidence for multimetric states of SL-AP-B over the pH range 2-9, with maximum aggregation at pH 4.5-5, and a slow disaggregation when the pH is adjusted to 8-9. The presence of multiple conformers of SL-AP-B and its tendency to aggregate render it unsuitable for high-resolution NMR structural studies of the isolated ligand, but the retention of activity may make it useful for studies of the sodium-channel-bound form of the molecule.

Solution structure of human neuropeptide Y.

The three-dimensional structure of synthetic human neuropeptide Y in aqueous solution at pH 3.2 and 37 degrees C was determined from two-dimensional 1H NMR data recorded at 600 MHz. A restraint set consisting of 440 interproton distance restraints inferred from NOEs and 11 backbone and 4 side-chain dihedral angle restraints derived from spin-spin coupling constants was used as input for distance geometry calculations on DIANA and simulated annealing and restrained energy minimization in X-PLOR. The final set of 26 structures is well defined in the region of residues 11-36, with a mean pairwise rmsd of 0.51 A for the backbone heavy atoms (N, C alpha and C) and 1.34 A for all heavy atoms. Residues 13-36 form an amphipathic alpha-helix. The N-terminal 10 residues are poorly defined relative to the helical region, although some elements of local structure are apparent. At least one of the three prolines in the N-terminal region co-exists in both cis and trans conformations. An additional set of 24 distances was interpreted as intermolecular distances within a dimer. A combination of distance geometry and restrained simulated annealing yielded a model of the dimer having antiparallel packing of two helical units, whose hydrophobic faces form a well-defined core. Sedimentation equilibrium experiments confirm the observation that neuropeptide Y associates to form dimers and higher aggregates under the conditions of the NMR experiments. Our results therefore support the structural features reported for porcine neuropeptide Y [Cowley, D.J. et al. (1992) Eur. J. Biochem., 205, 1099-1106] rather than the 'aPP' fold described previously for human neuropeptide Y [Darbon, H. et al. (1992) Eur. J. Biochem., 209, 765-771].

A test for linkage and association in general pedigrees: the pedigree disequilibrium test.

Family-based tests of linkage disequilibrium typically are based on nuclear-family data including affected individuals and their parents or their unaffected siblings. A limitation of such tests is that they generally are not valid tests of association when data from related nuclear families from larger pedigrees are used. Standard methods require selection of a single nuclear family from any extended pedigrees when testing for linkage disequilibrium. Often data are available for larger pedigrees, and it would be desirable to have a valid test of linkage disequilibrium that can use all potentially informative data. In this study, we present the pedigree disequilibrium test (PDT) for analysis of linkage disequilibrium in general pedigrees. The PDT can use data from related nuclear families from extended pedigrees and is valid even when there is population substructure. Using computer simulations, we demonstrated validity of the test when the asymptotic distribution is used to assess the significance, and examined statistical power. Power simulations demonstrate that, when extended pedigree data are available, substantial gains in power can be attained by use of the PDT rather than existing methods that use only a subset of the data. Furthermore, the PDT remains more powerful even when there is misclassification of unaffected individuals. Our simulations suggest that there may be advantages to using the PDT even if the data consist of independent families without extended family information. Thus, the PDT provides a general test of linkage disequilibrium that can be widely applied to different data structures.

Three-dimensional structure in solution of the polypeptide cardiac stimulant anthopleurin-A.

The three-dimensional structure in aqueous solution of the 49-residue polypeptide anthopleurin-A (AP-A), from the sea anemone Anthopleura xanthogrammica, has been determined from 1H NMR data. A restraint set consisting of 411 interproton distance restraints inferred from NOEs and 19 backbone and 13 side chain dihedral angle restraints from spin-spin coupling constants, as well as 15 lower bound restraints based on the absence of NOEs in the spectra, was used as input for distance geometry calculations in DIANA and simulated annealing and restrained energy minimization in X-PLOR. Stereospecific assignments for 12 beta-methylene pairs were also included. The final set of 20 structures had mean pairwise rms differences over the whole molecule of 2.04 A for the backbone heavy atoms (N, C alpha, and C) and 2.59 A for all heavy atoms. For the well-defined region encompassing residues 2-7 and 17-49, the corresponding values were 0.82 and 1.27 A, respectively. AP-A adopts a compact structure consisting of four short strands of antiparallel beta-sheet (residues 2-4, 20-23, 34-37, and 45-48) connected by three loops. The first loop commences with a type I beta-turn which includes two important Asp residues; this loop is the least well-defined region of the protein, although a beta-turn involving residues 13-16 is observed in nearly half the structures. The loop linking the second and third strands is constrained by the 29-47 disulfide bond and contains two well-defined beta-turns, while the third loop contains the Gly40-Pro41 sequence, which has been identified previously as the site of cis-trans isomerism. The carboxylate group of Asp7 is close to the epsilon-ammonium group of Lys37, suggesting that they may form a salt bridge. A pH titration monitored by 2D NMR supports this by showing that Asp7 has a low pKa. It is proposed that this region of the molecule and the nearby residues Asp9 and His39 form part of the molecular surface which interacts with the mammalian cardiac sodium channel.

Two tests of association for a susceptibility locus for families of variable size: an example using two sampling strategies.

A two-stage approach was used to analyze Problem 2 simulated data from Genetic Analysis Workshop 11. In the first stage, we tested for linkage with the Haseman-Elston test in SIBPAL. Markers that were significant in the first stage were followed up with two types of association tests. These association tests differ in the type of family information used: 1) parental transmissions to affected children or 2) differences in marker allele frequencies between affected and unaffected siblings. We also explored how the conclusions changed when different sampling strategies were used. Of particular interest was whether the entire data set should be used to test for both linkage and association or whether the data set should be halved to allow for replication of the initial association results.

Solution structure of a polypeptide from the N terminus of the HIV protein Nef.

Nef is a 27 kDa myristylated phosphoprotein expressed early in infection by HIV. The N terminus of Nef is thought to play a vital role in the functions of this protein through its interactions with membrane structures. The solution structure of a 25-residue polypeptide corresponding to the N terminus of Nef (Nef1-25) has been investigated by 1H NMR spectroscopy. In aqueous solution at pH 4.8 and 281 K, this peptide underwent conformational averaging, with Pro13 existing in cis and trans conformations in nearly equal proportions. In methanol solution, however, the peptide adopted a well-defined alpha-helical structure from residues 6 to 22, with the N- and C-terminal regions having a less ordered structure. On the basis of a comparison of chemical shifts and NOEs, it appeared that this helical structure was maintained in aqueous trifluoroethanol (50% v/v) and to a lesser extent in a solution of SDS micelles. When the N-acetyl group was replaced by either an N-myristyl or a free ammonium group, there was little effect on the three-dimensional structure of the peptide in methanol; deamidation of the C terminus also had no effect on the structure in methanol. In water, the myristylated peptide aggregated. The similarity between the sequences of Nef1-25 and melittin is reflected in the similar structures of the two molecules, although the N-terminal helix of melittin is more defined. This similarity in structure raises the possibility that Nef1-25 not only interacts with membranes but also may be capable of disrupting them and causing cell lysis. This type of interaction could contribute at least in part to the killing of bystander cells in lymphoid tissues during HIV infection.

Genetic inheritance of gene expression in human cell lines.

Combining genetic inheritance information, for both molecular profiles and complex traits, is a promising strategy not only for detecting quantitative trait loci (QTLs) for complex traits but for understanding which genes, pathways, and biological processes are also under the influence of a given QTL. As a primary step in determining the feasibility of such an approach in humans, we present the largest survey to date, to our knowledge, of the heritability of gene-expression traits in segregating human populations. In particular, we measured expression for 23,499 genes in lymphoblastoid cell lines for members of 15 Centre d'Etude du Polymorphisme Humain (CEPH) families. Of the total set of genes, 2,340 were found to be expressed, of which 31% had significant heritability when a false-discovery rate of 0.05 was used. QTLs were detected for 33 genes on the basis of at least one P value <.000005. Of these, 13 genes possessed a QTL within 5 Mb of their physical location. Hierarchical clustering was performed on the basis of both Pearson correlation of gene expression and genetic correlation. Both reflected biologically relevant activity taking place in the lymphoblastoid cell lines, with greater coherency represented in Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathways than in Gene Ontology database pathways. However, more pathway coherence was observed in KEGG pathways when clustering was based on genetic correlation than when clustering was based on Pearson correlation. As more expression data in segregating populations are generated, viewing clusters or networks based on genetic correlation measures and shared QTLs will offer potentially novel insights into the relationship among genes that may underlie complex traits.

Molecular evidence of greater selective pressure for drug resistance exerted by the long-acting antifolate Pyrimethamine/Sulfadoxine compared with the shorter-acting chlorproguanil/dapsone on Kenyan Plasmodium falciparum.

Pyrimethamine (PM) plus sulfadoxine (SD) is the last remaining affordable drug for treating uncomplicated malaria in Africa. The selective pressure exerted by the slowly eliminated combination PM/SD was compared with that exerted by the more rapidly eliminated combination chlorproguanil/dapsone (CPG/Dap) on Kenyan Plasmodium falciparum. Point mutations were analyzed in dihydrofolate reductase and dihydropteroate synthase and in the genetic diversity of 3 genes in isolates collected before and after CPG/Dap and PM/SD treatments. PM/SD was associated strongly with the disappearance of fully drug-sensitive parasites and with a significant increase in the prevalence of resistant parasites in subsequent parasitemias. However, this was not a characteristic of treatment with CPG/Dap. Moreover, most of the patients who returned with recrudescent infections were in the PM/SD-treated group. The data predict a longer useful therapeutic life for CPG/Dap than for PM/SD, and, thus, CPG/Dap is a preferable alternative for treatment of chloroquine-resistant falciparum malaria in sub-Saharan Africa.