Discriminating and locating character covariance: an application of permutation tail probability (PTP) analyses

The ability of permutation tail probability (PTP) analyses to discriminate between character covariance and noise is investigated with both hypothetical and published data sets. PTP is shown to be a powerful tool, not only for detecting character covariance, but also for locating that covariance on trees. PTP is especially useful for evaluating DNA sequence data that may have a high level of homoplasy. A three-step PTP procedure for locating covaried characters is presented.     

When Are Random Data Not Random,or Is the PTP Test Useful?

Recently, empirical evidence was presented that the permutation tail probability (PTP) test has extremely low discriminatory power when assessing character covariance in phylogenetic data based on bootstrap measures of confidence. Here we are concerned with the problem of using one statistical approach, especially when applied to empirical data, to judge the performance of another. Applying an appropriate statistical approach, we statistically demonstrated that the PTP test is extremely weak in detecting the absence of character covariation. In addition, we show that PTP is highly dependent on the number of terminals and the proportion of character states in phylogenetic matrices. In conclusion, we advocate the use of simulation studies when testing the performance of statistical tools applied to phylogenetic data.     

PTP is Meaningless,T-PTP is Contradictory: A Reply to Trueman

Abstract — The T-PTP test for monophyly can attribute significance to entirely unsupported groups and even to both of two contradictory alternatives. The method of evaluating “support” after replacing selected groups of terminals with reconstructed ancestors has similar drawbacks. The proposed placement of Onychophora among Arthropoda is unsupported by 12S data, and strongly refuted by other evidence. Attempts to justify T-PTP on Popperian grounds rest entirely on misunderstanding Popper's ideas. The PTP test assesses neither Popperian corroboration nor statistical confidence of phylogenetic conclusions. It can attribute high significance to data that support no resolved grouping. Efforts to salvage PTP by proposing new interpretations share the weakness that none of the proposed interpretations generally holds. None of these methods seems useful in phylogenetic     

Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects

We report the complete sequence of a paralogous copy of elongation factor-1 alpha (EF-1 alpha) in the honeybee, Apis mellifera (Hymenoptera: Apidae). This copy differs from a previously described copy in the positions of five introns and in 25% of the nucleotide sites in the coding regions. The existence of two paralogous copies of EF-1 alpha in Drosophila and Apis suggests that two copies of EF-1 alpha may be widespread in the holometabolous insect orders. To distinguish between a single, ancient gene duplication and parallel, independent fly and bee gene duplications, we performed a phylogenetic analysis of hexapod EF-1 alpha sequences. Unweighted parsimony analysis of nucleotide sequences suggests an ancient gene duplication event, whereas weighted parsimony analysis of nucleotides and unweighted parsimony analysis of amino acids suggests the contrary: that EF-1 alpha underwent parallel gene duplications in the Diptera and the Hymenoptera. The hypothesis of parallel gene duplication is supported both by congruence among nucleotide and amino acid data sets and by topology-dependent permutation tail probability (T-PTP) tests. The resulting tree topologies are also congruent with current views on the relationships among the holometabolous orders included in this study (Diptera, Hymenoptera, and Lepidoptera). More sequences, from diverse orders of holometabolous insects, will be needed to more accurately assess the historical patterns of gene duplication in EF-1 alpha.      

Molecular phylogeny ofForsythia (Oleaceae) based on chloroplast DNA variation

Phylogenetic relationships of ten wild species and several cultivars ofForsythia were reconstructed based on the chloroplast (cp) DNA variation. A total of 216 cpDNA variants, 44 of which were potentially phylogenetically informative, was detected using 24 restriction endonucleases. Phylogenetic analysis usingFontanesia andAbeliophyllum as outgroups revealed four well defined species groups in the genus: 1)F. suspensa, 2)F. europaea — F. giraldiana, 3)F. ovata — F. japonica — F. viridissima, and 4)F. koreana — F. manshurica — F. saxatilis. The amount of support for each monophyletic group was evaluated by various methods including character number, decay analysis, parsimony bootstrapping, Neighbour-Joining (NJ) — bootstrapping, NJ-jackknifing, and the topology-dependent permutation tail probability (T-PTP) test. The data do not support the hybrid origin ofF. intermedia fromF. suspensa andF. viridissima. The disjunctly distributed European species,F. europaea, was identified as a sister species of the ChineseF. giraldiana and it was probably derived through recent long distance dispersal.     

Subcellular Partitioning of Protein Tyrosine Phosphatase 1B to the Endoplasmic Reticulum and Mitochondria Depends Sensitively on the Composition of Its Tail Anchor

The canonical protein tyrosine phosphatase PTP1B is an important regulator of diverse cellular signaling networks. PTP1B has long been thought to exert its influence solely from its perch on the endoplasmic reticulum (ER); however, an additional subpopulation of PTP1B has recently been detected in mitochondria extracted from rat brain tissue. Here, we show that PTP1B’s mitochondrial localization is general (observed across diverse mammalian cell lines) and sensitively dependent on the transmembrane domain length, C-terminal charge and hydropathy of its short (≤35 amino acid) tail anchor. Our electron microscopy of specific DAB precipitation revealed that PTP1B localizes via its tail anchor to the outer mitochondrial membrane (OMM), with fluorescence lifetime imaging microscopy establishing that this OMM pool contributes to the previously reported cytoplasmic interaction of PTP1B with endocytosed epidermal growth factor receptor. We additionally examined the mechanism of PTP1B’s insertion into the ER membrane through heterologous expression of PTP1B’s tail anchor in wild-type yeast and yeast mutants of major conserved ER insertion pathways: In none of these yeast strains was ER targeting significantly impeded, providing in vivo support for the hypothesis of spontaneous membrane insertion (as previously demonstrated in vitro). Further functional elucidation of the newly recognized mitochondrial pool of PTP1B will likely be important for understanding its complex roles in cellular responses to external stimuli, cell proliferation and diseased states.     

Residues distant from the active site influence protein-tyrosine phosphatase 1B inhibitor binding

Regions of protein-tyrosine phosphatase (PTP) 1B that are distant from the active site yet affect inhibitor binding were identified by a novel library screen. This screen was based on the observation that expression of v-Src in yeast leads to lethality, which can be rescued by the coexpression of PTP1B. However, this rescue is lost when yeast are grown in the presence of PTP1B inhibitors. To identify regions of PTP1B (amino acids 1-400, catalytic domain plus 80-amino acid C-terminal tail) that can affect the binding of the difluoromethyl phosphonate (DFMP) inhibitor 7-bromo-6-difluoromethylphosphonate 3-naphthalenenitrile, a library coexpressing PTP1B mutants and v-Src was generated, and the ability of yeast to grow in the presence of the inhibitor was evaluated. PTP1B inhibitor-resistant mutations were found to concentrate on helix alpha7 and its surrounding region, but not in the active site. No resistant amino acid substitutions were found to occur in the C-terminal tail, suggesting that this region has little effect on active-site inhibitor binding. An in-depth characterization of a resistant substitution localizing to region alpha7 (S295F) revealed that this change minimally affected enzyme catalytic activity, but significantly reduced the potency of a panel of structurally diverse DFMP PTP1B inhibitors. This loss of inhibitor potency was found to be due to the difluoro moiety of these inhibitors because only the difluoro inhibitors were shifted. For example, the inhibitor potency of a monofluorinated or non-fluorinated analog of one of these DFMP inhibitors was only minimally affected. Using this type of library screen, which can scan the nearly full-length PTP1B sequence (catalytic domain and C-terminal tail) for effects on inhibitor binding, we have been able to identify novel regions of PTP1B that specifically affect the binding of DFMP inhibitors.     

Characterization of the C-terminal ER membrane anchor of PTP1B

The tyrosine phosphatase PTP1B is an important regulator of cell function. In living cells PTP1B activity is restricted to the vicinity of the endoplasmic reticulum (ER) by post-translational C-terminal attachment of PTP1B to the ER membrane network. In our study we investigated the membrane anchor of PTP1B by use of EGFP fusion proteins. We demonstrate that the membrane anchor of PTP1B cannot be narrowed down to a unique amino acid sequence with a defined start and stop point but rather is moveable within several amino acids. Removal of up to seven amino acids from the C-terminus, as well as exchange of single amino acids in the putative transmembrane sequence did not influence subcellular localization of PTP1B. With the method of bimolecular fluorescence complementation we could demonstrate dimerization of PTP1B in vivo. Homodimerization was, in contrast to other tail-anchored proteins, not dependent on the membrane anchor. Our data demonstrate that the C-terminal membrane anchor of PTP1B is formed by a combination of a single stretch transmembrane domain (TMD) followed by a tail. TMD and tail length are variable and there are no sequence-specific features. Our data for PTP1B are consistent with a concept that explains the ER membrane anchor of tail-anchored proteins as a physicochemical structure.     

Phylogenetic signal in AFLP data sets

AFLP markers provide a potential source of phylogenetic information for molecular systematic studies. However, there are properties of restriction fragment data that limit phylogenetic interpretation of AFLPs. These are (a) possible nonindependence of fragments, (b) problems of homology assignment of fragments, (c) asymmetry in the probability of losing and gaining fragments, and (d) problems in distinguishing heterozygote from homozygote bands. In the present study, AFLP data sets of Lactuca s.l. were examined for the presence of phylogenetic signal. An indication of this signal was provided by carrying out tree length distribution skewness (g1) tests, permutation tail probability (PTP) tests, and relative apparent synapomorphy analysis (RASA). A measure of the support for internal branches in the optimal parsimony tree (MPT) was made using bootstrap, jackknife, and decay analysis. Finally, the extent of congruence in MPTs for AFLP and internal transcribed spacer (ITS)-1 data sets for the same taxa was made using the partition homogeneity test (PHT) and the Templeton test. These analytical studies suggested the presence of phylogenetic signal in the AFLP data sets, although some incongruence was found between AFLP and ITS MPTs. An extensive literature survey undertaken indicated that authors report a general congruence of AFLP and ITS tree topologies across a wide range of taxonomic groups, suggesting that the present results and conclusions have a general bearing. In these earlier studies and those for Lactuca s.l., AFLP markers have been found to be informative at somewhat lower taxonomic levels than ITS sequences. Tentative estimates are suggested for the levels of ITS sequence divergence over which AFLP profiles are likely to be phylogenetically informative.     

Permutation P-values should never be zero: calculating exact P-values when permutations are randomly drawn

Permutation tests are amongst the most commonly used statistical tools in modern genomic research, a process by which p-values are attached to a test statistic by randomly permuting the sample or gene labels. Yet permutation p-values published in the genomic literature are often computed incorrectly, understated by about 1/m, where m is the number of permutations. The same is often true in the more general situation when Monte Carlo simulation is used to assign p-values. Although the p-value understatement is usually small in absolute terms, the implications can be serious in a multiple testing context. The understatement arises from the intuitive but mistaken idea of using permutation to estimate the tail probability of the test statistic. We argue instead that permutation should be viewed as generating an exact discrete null distribution. The relevant literature, some of which is likely to have been relatively inaccessible to the genomic community, is reviewed and summarized. A computation strategy is developed for exact p-values when permutations are randomly drawn. The strategy is valid for any number of permutations and samples. Some simple recommendations are made for the implementation of permutation tests in practice.     

The future of phylogeny reconstruction

A new approach to phylogenetic analysis, parsimony jackknifing, uses simple parsimony calculations combined with resampling of characters to arrive at a tree comprising well-supported groups. This is usually much the same as the consensus of most-parsimonious trees found from extensive multiple-tree calculations, but the new method is thousands of times faster, allowing analysis of much larger data matrices, and also provides information on the strength of support for different groups. Jackknife frequencies provide a more reliable assessment of support than do alternative methods, notably "confidence probability" (CP) and T-PTP testing.     

COULD A CLADOGRAM THIS SHORT HAVE ARISEN BY CHANCE ALONE?: ON PERMUTATION TESTS FOR CLADISTIC STRUCTURE

Abstract Absolute criteria for evaluating cladistic analyses are useful, not only because cladistic algorithms impose structure, but also because applications of cladistic results demand some assessment of the degree of corroboration of the cladogram. Here, a means of quantitative evaluation is presented based on tree length. The length of the most-parsimonious tree reflects the degree to which the observed characters co-vary such that a single tree topology can explain shared character states among the taxa. This “cladistic covariation” can be quantified by comparing the length of the most parsimonious tree for the observed data set to that found for data sets with random covariation of characters. A random data set is defined as one in which the original number of characters and their character states are maintained, but for each character, the states are randomly reassigned to the taxa. The cladistic permutation tail probability, PTP, is defined as the estimate of the proportion of times that a tree can be found as short or shorter than the original tree. Significant cladistic covariation exists if the PTP is less than a prescribed value, for example, 0.05. In case studies based on molecular and morphological data sets, application of the PTP shows that:

• 1 In the comparison of four different molecular data sets for orders of mammals, the sequence data set for alpha hemoglobin does not have significant cladistic covariation, while that for alpha crystallin is highly significant. However, when each data set was reduced to the 11 common taxa in order to standardize comparison, reduced levels of cladistic covariation, with no clear superiority of the alpha crystallin data, were found. Morphological data for these 11 taxa had a highly significant PTP, producing a tree roughly congruent with those for the three molecular sets with marginal or significant PTP values. Merging of all data sets, with the exclusion of the poorly structured alpha hemoglobin data, produced a data set with a significant PTP, and provides an estimate of the phylogenetic relationships among these 11 orders of mammals.
• 2 In an analysis of lactalbumin and lysozyme DNA sequence data for four taxa, purine-pyrimidine coding yields a data set with significant cladistic covariation, while other codings fail. The data for codon position 3 taken alone exhibit the strongest cladistic covariation.
• 3 A data set based on flavonoids in taxa of Polygonum initially yields a significant PTP; however, deletion of identically scored taxa leaves no significant cladistic covariation.
• 4 For mitochondrial DNA data on population genome types for four species of the crested newt, there is significant cladistic covariation for the set of all genome types, and among the five mtDNA genome types within one of the species. However, a conditional PTP test that assumes species monophyly shows that no significant cladistic covariation exists among the fur species for these data.
• 5 In an application of the test to a group of freshwater insects, as preliminary to biological monitoring, individual subsets of the taxonomic data representing larval, pupal, and adult stages had non-significant PTPs, while the complete data set showed significant cladistic structure.

     

Mitochondrial permeability transition and cytochrome c release induced by selenite

Cytochrome c release and mitochondrial permeability transition (MPT) play important roles in apoptosis. In this study, we found that selenium, an essential trace element, induced mitochondrial membrane potential (Delta psi(m)) loss, swelling, and cytochrome c release in isolated mitochondria. All of the above observations were blocked by cyclosporin A (CsA), which is a specific inhibitor to permeability transition pore (PTP), indicating selenite-induced mitochondrial changes were mediated through the opening of PTP. In physiological concentration, selenite could induce mitochondria at low-conductance PTP 'open' probability, which is correlated to regulate the physiological function, whereas in toxic concentration, induce mitochondria at high-conductance PTP 'open' probability and rapidly undergo a process of osmotic swelling following diffusion toward matrix as for inducer (Ca(2+)/P(i)). Selenite also induced other mitochondrial marker enzymes including monoamine oxidase (MAO) and mitochondria aspartate aminotransferase (mAST). Oligomycin inhibited the selenite-induced cytochrome c release and Delta psi(m) loss, showing that F(0)F(1)-ATPase was important in selenite or Ca(2+)/P(i)-induced MPT.     

Meta-analysis of character utility and phylogenetic information content in cladistic studies of ammonoids

While previous workers have argued persuasively that ammonoid workers should use cladistic approaches to reconstruct phylogeny, relatively few cladistic studies have been published to date. An essential yet challenging part of cladistic analysis is the selection of characters. Are certain types of characters more likely to show homoplasy? Are certain aspects of shell anatomy more likely to contain phylogenetically informative characters? Are datasets with more characters inherently better? To answer these questions, a meta-analysis of character data from published ammonoid phylogenies was performed. I compiled 14 datasets, published between 1989 and 2007, representing parsimony-based phylogenetic analyses of ammonoids. These studies defined a combined total of 323 characters, which were grouped into categories reflecting different aspects of anatomy: shell size and shape, ornament, suture, early ontogeny, body chamber and apertural modifications. Tree searches were re-run to determine overall tree statistics, parsimony permutation tail probability (PTP) tests were calculated to assess the phylogenetic information content of the matrices, and retention and rescaled consistency indices for each character were calculated. My analyses revealed that studies with higher character/taxon ratios did not necessarily produce trees with more information content and less homoplasy, as measured by retention or rescaled consistency indices, because additional characters were often parsimony-uninformative. Rather, studies with relatively few characters could produce high-quality trees if the characters were well-chosen and character states carefully defined. Characters related to the body chamber and adult aperture typically had retention indices of either 0 or 1, rarely in between, indicating that they either worked perfectly or not at all. Suture characters tended to have higher indices than shell shape or ornament characters, suggesting more phylogenetic information and less homoplasy in the suture line than in shell traits. These results should aid in the selection of characters for future cladistic studies of ammonoids.     

Rational design,synthesis, and structure–activity relationships of 5-amino-1H-pyrazole-4-carboxylic acid derivatives as protein tyrosine phosphatase 1B inhibitors

A series of novel amino-carboxylic based pyrazole as protein tyrosine phosphatase 1B (PTP1B) inhibitors were designed on the basis of structure-based pharmacophore model and molecular docking. Compounds containing different hydrophobic tail (1,2-diphenyl ethanone, oxdiadizole and dibenzyl amines) were synthesized and evaluated in PTP1B enzymatic assay. Structure–activity relationship based optimization resulted in identification of several potent, metabolically stable and cell permeable PTP1B inhibitors.     

Tyrosine phosphorylation, thiol status, and protein tyrosine phosphatase in rat epididymal spermatozoa

Sperm thiol oxidation and the ability to undergo protein tyrosine phosphorylation are associated with the acquisition of sperm motility and fertilizing ability during passage of spermatozoa through the epididymis. Phosphotyrosine levels in various cells are controlled by tyrosine kinase versus phosphatase, with the latter known to be inhibited by oxidation. In the present paper we examine whether changes in thiol status during sperm maturation affect rat sperm protein phosphotyrosine levels and protein phosphotyrosine phosphatase (PTP) activity. Tyrosine phosphorylation, as demonstrated by immunoblotting (IB), was significantly increased in several sperm tail proteins during maturation in the epididymis. Sperm thiol oxidation with diamide enhanced tail protein phosphorylation; reduction of disulfides with dithiothreitol diminished phosphorylation. In the sperm head, a moderate increase in tyrosine phosphorylation was accompanied by altered localization of phosphotyrosine proteins during maturation. Blocking of thiols and PTP activity with N-ethylmaleimide led to increased tyrosine phosphorylation of protamine in caput sperm heads. Several PTP bands were identified by IB. In the caput spermatozoa, a prominent level of the 50 kDa band was present, whereas in the cauda spermatozoa a very low level of the 50 kDa band was found. PTP activity, measured by using p-nitrophenyl phosphate as a substrate, was significantly higher in the caput spermatozoa (high thiol content) than in the cauda spermatozoa (low thiol content). Our results show that PTP activity is correlated with sperm thiol status and suggest that tyrosine phosphorylation of sperm proteins during sperm maturation is promoted by thiol oxidation and diminished PTP.     

Ligand-selective modulation of the permeability transition pore by arginine modification. Opposing effects of p-hydroxyphenylglyoxal and phenylglyoxal.

Chemical modification of mitochondria with the arginine-specific reagents phenylglyoxal (PGO) and 2,3-butanedione (BAD) decreases the Ca(2+) sensitivity of the permeability transition pore (PTP) and stabilizes it in the closed conformation (Eriksson, O., Fontaine, E., and Bernardi, P. (1998) J. Biol. Chem. 273, 12669-12674). Unexpectedly, modification of mitochondria with the arginine-specific reagent p-hydroxyphenylglyoxal (OH-PGO) resulted instead in PTP opening. Sequential modification with OH-PGO and PGO (or BAD) revealed that the effects on the PTP depended on the order of the additions. PTP opening was observed when OH-PGO preceded, and PTP closing was observed when OH-PGO followed, the addition of PGO (or BAD). The differential effects of OH-PGO and PGO on the PTP open probability (i) were not modified by the conformation-specific ligands of the adenine nucleotide translocase bongkrekate and atractylate; and (ii) were also observed in de-energized mitochondria, indicating that the effect is exerted directly on the PTP. OH-PGO dramatically sensitized PTP opening, which was triggered by depolarization even in the presence of EGTA. These data show that arginine modification modulates the PTP conformation in a ligand-selective fashion and suggest that the effects of OH-PGO, PGO, and BAD are mediated by the same arginine residues. We analyzed the structure of the arginine adducts by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry using a test peptide and N-acetylarginine. The results indicate that both OH-PGO and PGO react with arginine at a stoichiometry of 2:1 and form stable adducts that may be feasible to identify the PTP at the molecular level.     

Reduction of PTP1B by RNAi upregulates the activity of insulin controlled fatty acid synthase promoter

Metabolic deregulation accompanying type II diabetes is characterized by insulin resistance in peripheral tissues (liver, muscle, and adipose), mediated by impairments in insulin receptor (IR) signaling. Protein tyrosine phosphatase 1B (PTP1B) has been shown to be a negative regulator of IR autophosphorylation and thus has been considered as a major therapeutic target for the treatment of type II diabetes. We use RNA interference technique to downregulate PTP1B expression in hepatoma cell line. A secretory HBV s-antigen was introduced as reporter and driven by mouse fatty acid synthase promoter, which is positively controlled by insulin signaling. Liver-targeted hydrodynamic injection in tail vein was introduced to transfer siRNA (or siRNA expression vector) and reporter plasmid into mouse liver. On fasted/refed and glucose stimulation condition, the HBV s-antigen in sera in RNAi group was higher than that in the negative group. Our results provided evidence that upregulation of insulin signaling by reducing PTP1B liver with RNAi can be a potent diabetes treatment method.     

Nitric oxide and differential effects of ATP on mitochondrial permeability transition.

The mitochondrial permeability transition pore (PTP) undergoes a calcium-dependent transition (MPT) that disrupts membrane potential and releases apoptogenic proteins. Because PTP opening is enhanced by oxidation of thiols at the so-called "S-site," we hypothesized that nitrogen monoxide (NO*) could enhance the open probability of the PTP, e.g., by S-nitrosylation or S-thiolation. At low NO donor concentrations (1 to 20 microM), PTP opening in succinate-energized liver mitochondria at nonlimiting calcium was delayed or unaffected, while it was accelerated by NO donors at 20 to 100 microM. At low donor concentrations, PTP opening was facilitated twofold by adenosine triphosphate (ATP), which normally delays PTP opening. Among NO donors, the oxatriazole GEA 3162, with an activation constant (Ka) of 1.9 microM at 500 microM ATP was more effective at enhancing pore transition than SIN-1 or SNAP. NO donor effects were superseded by diamide, which induces disulfide formation, but independent of SH-adduct formation by alkylation. NO-related changes in PTP function were accompanied by protein mixed disulfide formation, inhibited by dithiothreitol (DTT), and reversed by DTT after donor addition. PTP opening was stimulated in the presence of ATP by L-arginine-dependent NO production, i.e., mitochondrial NOS activity. ATP-facilitated pore opening was sensitive to atractyloside and depended on nucleotide interactions but not on hydrolysis, because specific nonhydrolyzable ATP analogs accelerated pore opening. These data indicate NO can influence pore transition by oxidation of thiols that produce conformational changes governing the ATP interaction at the adenine nucleotide transporter.