Synthesis and properties of a conformationally restricted spin-labeled analog of ATP and its interaction with myosin and skeletal muscle.

The synthesis is described of a spin-labeled analog of ATP, 2',3'-O-(1-oxy-2,2,6,6-tetramethyl-4-piperidylidene)adenosine 5'-triphosphate (SL-ATP). The spin-label moiety is attached by two bonds to the ribose ring as a spiroketal and hence has restricted conformational mobility relative to the ribose moiety of ATP. The synthesis proceeds via an acid-catalyzed addition of adenosine 5'-monophosphate to 1-acetoxy-4-methoxy-2,2,6,6-tetramethyl-1,2,5,6-tetrahydropyridine in acetonitrile. The spiroketal product is pyrophosphorylated, and alkaline hydrolysis with concomitant aerial oxidation gives the required product. The spin-labeled moiety probably takes up two rapidly interconverting conformations with respect to the ribose ring on the basis of the 1H NMR spectra of its precursors and related uridine derivatives [Alessi et al. (1991) J. Chem. Soc., Perkin Trans.1,2243-2247]. SL-ATP is a substrate for myosin and actomyosin with similar kinetic parameters to ATP during triphosphatase activity. SL-ATP supports muscle contraction and permits relaxation of permeabilized rabbit skeletal muscle fibers. SL-ADP is a substrate for yeast 3-phosphoglycerate kinase, thus permitting regeneration of SL-ATP from SL-ADP within muscle fibers. Electron paramagnetic resonance (EPR) studies of SL-ADP bound to myosin filaments and to myofibrils show a degree of nanosecond motion independent of that of the protein, which may be due to conformational flexibility of the ribose moiety of ATP bound to myosin's active site. This nanosecond motion is more restricted in myofibrils than in myosin filaments, suggesting that the binding of actin affects the ribose binding site in myosin. EPR studies on SL-ADP bound to rigor cross-bridges in muscle fiber bundles showed the nucleotide to be highly oriented with respect to the fiber axis.     

The phenotype of some late-flowering mutants is enhanced by a locus on chromosome 5 that is not effective in the Landsberg erecta wild-type

Late-flowering mutants that have been described in ecotypes other than Landsberg erecta (Ler) have been found to be dominant alleles of the FRI locus located on chromosome 4, which determines lateness in many very late ecotypes. The extreme lateness of dominant FRI alleles depends on dominant alleles at the FLC locus that maps on the top of chromosome 5. FLC alleles with this effect have been found in all ecotypes tested (Col, Ws, S96, Est and Li) except Ler. Most likely the same locus confers lateness to the luminidependens (ld) mutant. Genotypes with a dominant FRI allele and the monogenic recessive ld mutant are only slightly later with recessive Ler alleles at the FLC locus. Genotypes where the dominant FLC alleles are combined with FRI or with the ld mutant, are strongly responsive to vernalization, which is much less effective in the FLC-Ler background.     

Spine and dine: A key defensive trait promotes ecological success in spiny ants

A key focus of ecologists is explaining the origin and maintenance of morphological diversity and its association with ecological success. We investigate potential benefits and costs of a common and varied morphological trait, cuticular spines, for foraging behavior, interspecific competition, and predator–prey interactions in naturally co‐occurring spiny ants (Hymenoptera: Formicidae: Polyrhachis) in an experimental setting. We expect that a defensive trait like spines might be associated with more conspicuous foraging, a greater number of workers sent out to forage, and potentially increased competitive ability. Alternatively, consistent with the ecological trade‐off hypothesis, we expect that investment in spines for antipredator defense might be negatively correlated with these other ecological traits. We find little evidence for any costs to ecological traits, instead finding that species with longer spines either outperform or do not differ from species with shorter spines for all tested metrics, including resource discovery rate and foraging effort as well as competitive ability and antipredator defense. Spines appear to confer broad antipredator benefits and serve as a form of defense with undetectable costs to key ecological abilities like resource foraging and competitive ability, providing an explanation for both the ecological success of the study genus and the large number of evolutionary origins of this trait across all ants. This study also provides a rare quantitative empirical test of ecological effects related to a morphological trait in ants.     

A retrospective study of DNA prevalence of porcine parvoviruses in Mexico and its relationship with porcine circovirus associated disease

Worldwide, many emerging porcine parvoviruses (PPVs) have been linked to porcine circovirus-2 (PCV2) associated disease (PCVAD), which includes post-weaning multi-systemic wasting syndrome (PMWS), PCV2-related reproductive failure (PCV2-RF), as well as other syndromes. To determine the DNA prevalence of PPVs and their relationship with PMWS and PCV2-RF in Mexico, 170 formalin-fixed paraffin-embedded tissues were selected from archival collections to detect PPVs using a nested polymerase chain reaction. The tissues were composed of 50 PMWS cases, 20 age-matched tissues from healthy pigs, 56 PCV2-related reproductive failure (PCV2⁺-RF) cases, and 44 PCV2^--RF cases. Overall, PPV2 and PPV6 were the most prevalent species (90.0% and 74.7%, respectively). In 8–11 week old pigs, the highest prevalence was for PPV6 and PPV3. Concerning reproductive failure, the PCV2-affected farms had a significantly higher prevalence for PPV6 (61.6%) and PPV5 (36.4%) than the PCV2-unaffected farms (35.0% and 5.0%, respectively). The concurrent infection rate was high, being significant for PPV2/PPV4 and PPV1/PPV5 within the PMWS cases and for PPV6/PPV5 among the PCV2⁺-RF tissues. PPV5 showed a significant relationship with PMWS, whereas PPV5 and PPV6 were significant for PCVAD. The prevalence and coinfection rate of PPVs in Mexico were markedly higher than that described in other countries, denoting that PPV5 and PPV6 might have a potential role in PCVAD in Mexico. It is concluded that it is likely that the density population of pigs in Mexico is contributing to high PPV inter-species and PCV2 coinfections which might lead to a different pathogenic outcome.     

Identification of somatic chromosomal abnormalities in hypothalamic hamartoma tissue at the GLI3 locus

Hypothalamic hamartomas (HH) are rare, benign congenital tumors associated with intractable epilepsy. Most cases are sporadic and nonsyndromic. Approximately 5% of HH cases are associated with Pallister-Hall syndrome (PHS), which is caused by haploinsufficiency of GLI3. We have investigated the possibility that HH pathogenesis in sporadic cases is due to a somatic (tumor-only) mutation in GLI3. We isolated genomic DNA from peripheral blood and surgically resected HH tissue in 55 patients with sporadic HH and intractable epilepsy. A genome-wide screen for loss of heterozygosity (LOH) and chromosomal abnormalities was performed with parallel analysis of blood and HH tissue with Affymetrix 10K SNP microarrays. Additionally, resequencing and fine mapping with SNP genotyping were completed for the GLI3 gene with comparisons between peripheral blood and HH tissue pairs. By analyzing chromosomal copy-number data for paired samples on the Affymetrix 10K array, we identified a somatic chromosomal abnormality on chromosome 7p in one HH tissue sample. Resequencing of GLI3 did not identify causative germline mutations but did identify LOH within the GLI3 gene in the HH tissue samples of three patients. Further genotyping of 28 SNPs within and surrounding GLI3 identified five additional patients exhibiting LOH. Together, these data provide evidence that the development of chromosomal abnormalities within GLI3 is associated with the pathogenesis of HH lesions in sporadic, nonsyndromic patients with HH and intractable epilepsy. Chromosomal abnormalities including the GLI3 locus were seen in 8 of 55 (15%) of the resected HH tissue samples. These somatic mutations appear to be highly variable.     

A fluorescent sensor of the phosphorylation state of nucleoside diphosphate kinase and its use to monitor nucleoside diphosphate concentrations in real time

A sensor for purine nucleoside diphosphates in solution based on nucleoside diphosphate kinase (NDPK) has been developed. A single cysteine was introduced into the protein and labeled with the environmentally sensitive fluorophore, N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide. The resultant molecule shows a 4-fold fluorescence increase when phosphorylated on His117; this phosphorylation is on the normal reaction pathway of the enzyme. The emission maximum of the phosphoenzyme is at 475 nm, with maximum excitation at 430 nm. The fluorescent phosphorylated NDPK is used to measure the amount of ADP and the unphosphorylated to measure ATP. The labeled protein is phosphorylated to > 90%, and the resultant molecule is stable on ice or can be stored at -80 degrees C. The fluorescence responds to the fraction of protein phosphorylated and so to the equilibrium between ADP plus NDPK approximately P and ATP plus NDPK. In effect, the sensor measures the ADP/ATP concentration ratio. The enzyme has a broad specificity for the purine of the nucleotides, so the sensor also can measure GDP/GTP ratios. The fluorescence and kinetic properties of the labeled protein are described. The binding rate constants of nucleotides are approximately 10(5) M(-1) s(-1), and the fluorescence change is at >200 s(-1) when the ADP concentration is >1 mM. Results are presented with two well-defined systems, namely, the kinetics of ADP release from myosin subfragment 1 and GDP release from the small G protein, human rho. The results obtained with this novel sensor agree with those from alternate methods and demonstrate the applicability for following micromolar changes in nucleoside diphosphate in real time.     

Elucidation of a PTS-carbohydrate chemotactic signal pathway in Escherichia coli using a time-resolved behavioral assay

Chemotaxis of Escherichia coli toward phosphotransferase systems (PTSs)-carbohydrates requires phosphoenolpyruvate-dependent PTSs as well as the chemotaxis response regulator CheY and its kinase, CheA. Responses initiated by flash photorelease of a PTS substrates D-glucose and its nonmetabolizable analog methyl alpha-D-glucopyranoside were measured with 33-ms time resolution using computer-assisted motion analysis. This, together with chemotactic mutants, has allowed us to map out and characterize the PTS chemotactic signal pathway. The responses were absent in mutants lacking the general PTS enzymes EI or HPr, elevated in PTS transport mutants, retarded in mutants lacking CheZ, a catalyst of CheY autodephosphorylation, and severely reduced in mutants with impaired methyl-accepting chemotaxis protein (MCP) signaling activity. Response kinetics were comparable to those triggered by MCP attractant ligands over most of the response range, the most rapid being 11.7 +/- 3.1 s-1. The response threshold was <10 nM for glucose. Responses to methyl alpha-D-glucopyranoside had a higher threshold, commensurate with a lower PTS affinity, but were otherwise kinetically indistinguishable. These facts provide evidence for a single pathway in which the PTS chemotactic signal is relayed rapidly to MCP-CheW-CheA signaling complexes that effect subsequent amplification and slower CheY dephosphorylation. The high sensitivity indicates that this signal is generated by transport-induced dephosphorylation of the PTS rather than phosphoenolpyruvate consumption.     

Support for the allotonic frequency hypothesis in an insectivorous bat community

The allotonic frequency hypothesis proposes that certain insectivorous bat species can prey upon moths that can hear bat echolocation calls by using echolocation frequencies outside the sensitivity range of moth ears. The hypothesis predicts that the peak frequencies of bat echolocation calls are correlated with the incidence of moths in the diets of these bats. The aim of this study was to test this prediction on a bat community dominated by bats using low duty cycle echolocation calls, i.e. aerial foraging, insectivorous species using frequency modulated calls. The community consisted of nine species, two molossids, Sauromys petrophillus and Tadarida aegyptiaca, five vespertilionids, Eptesicus capensis, Eptesicus hottentotus, Miniopteris schreibersii, Myotis tricolor, and Myotis lesueuri, one rhinolophid, Rhinolophus clivosus, and one nycterid, Nycteris thebaica. The insect fauna in the habitat used by the bat community was suited to the testing of the allotonic frequency hypothesis because more than 90% of the moths comprising the insect fauna were tympanate. These included Pyralidae (3.8%), Geometridae (44.9%), Notodontidae (3.8%), Arctiidae (4.6%), Lymantriidae (0.8%) and Noctuidae (32.4%). As predicted, peak echolocation frequency was correlated with the incidence of moths in the diets of these nine species (r=0.98, df=7, P<0.01). Furthermore, multivariate analysis revealed that echolocation frequency (t=9.91, n=129, P<0.001) was a better predictor of diet than forearm length (t=5.51, n=129, P<0.001) or wing area (t=-3.41, n=129, P<0.001). This suggests that the selection pressure exerted by moth hearing might have acted directly on call frequency and secondarily on body size and wing morphology, as part of the same adaptive complex. It is unlikely that dietary differences were due to temporal and spatial differences in the availability of prey because the pattern of differences in skull morphology of the nine species supported our dietary analyses. The skull morphology of a bat represents a historical record of the kind of diet it has become adapted to over its evolutionary history. These results suggest that prey defences may mediate other factors structuring bat communities, e.g. competition. Competition may be reduced for those species of bats that can circumvent prey defences.