Evolutionarily Divergent, Unstable Filamentous Actin Is Essential for Gliding Motility in Apicomplexan Parasites

Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility

Gene discovery in the Apicomplexa as revealed by EST sequencing and assembly of a comparative gene database. Genome Res

Large-scale EST sequencing projects for several important parasites within the phylum Apicomplexa were undertaken for the purpose of gene discovery. Included were several parasites of medical importance (Plasmodium falciparum, Toxoplasma gondii) and others of veterinary importance (Eimeria tenella, Sarcocystis neurona, and Neospora caninum). A total of 55,192 ESTs, deposited into dbEST/GenBank, were included in the analyses. The resulting sequences have been clustered into nonredundant gene assemblies and deposited into a relational database that supports a variety of sequence and text searches. This database has been used to compare the gene assemblies using BLAST similarity comparisons to the public protein databases to identify putative genes. Of these new entries, ∼15%-20% represent putative homologs with a conservative cutoff of p < 10 −9 , thus identifying many conserved genes that are likely to share common functions with other well-studied organisms. Gene assemblies were also used to identify strain polymorphisms, examine stage-specific expression, and identify gene families. An interesting class of genes that are confined to members of this phylum and not shared by plants, animals, or fungi, was identified. These genes likely mediate the novel biological features of members of the Apicomplexa and hence offer great potential for biological investigation and as possible therapeutic targets

Converter system nonlinear modelling and control for transmission applications - part II: CSC systems

The high-power self-commutated voltage-source converter (VSC) and current-source converter (CSC) are the key control devices in high-voltage direct current, flexible ac transmission systems, and distribution flexible ac transmission systems. To achieve the expected control objectives, suitable control strategies must be implemented based on the available devices, system models, and control techniques. The self-commutated ac/dc converters control the electrical power by generating controllable ac fundamental and dc average outputs. These controllable outputs are controlled by the conducting state combinations of the converter switching devices, driven by their gate signals. The gate signals are specified by fundamental parameters of frequency, amplitude, and phase angle. The converter system model for describing the relation between the system-state variables and the gate signal parameters is essential for the converter system control strategies. The companion paper (Part I) derives the state variable equations for the transmission systems using voltage-source-type converters. Part II is for the transmission systems using current-source-type converters. The self-commutated converter systems provide control flexibility of active and reactive powers, but their nonlinearity makes their control difficult. The linearized state equations using feedback linearization are presented to enable the controller design by using linear control theory

Structure of Py·Pu·PyDNA triple helices. Fourier transforms of fiber-type X-ray diffraction of single crystals

Well-formed hexagonal crystals of oligomeric DNA triple helices exhibit fiber-type x-ray diffraction patterns [cf., Liu et al. (1994) Nature Struct. Biol. 1, 11], which can be interpreted in terms of Fourier transforms of these helices. Precession photographs of a triplex formed of dA and dT chains show that it has 13 residues per turn. In contrast, a sequence containing the four natural bases A, G, C, and T has 12 residues per turn. In this sense the triple helices exhibit a sequence-dependent polymorphism, though both have C2'-endo sugar pucker and B rather than A conformation. New models are constructed, using constraints from x-ray diffraction, and Fourier transforms of the models are calculated. Good agreement in the amplitudes and positions of the calculated and observed diffraction intensities confirms the structures for both triple helices. These are the first stereochemically satisfactory DNA triple helices for which coordinates based on adequate experimental data were provided. Sequences for crystallization are designed to achieve unique base alignments and are screened for the presence of sharp bands on gel electrophoresis to assure the absence of multiple species caused by strand slippage. Despite intensive efforts to observe normal crystal diffraction by varying sequences and conditions, all crystals exhibited only fiber-type diffraction. We suggest that this behavior may be an intrinsic property of triple helices and discuss possible reasons for the results. Spectroscopic and chemical experiments establish that the oligonucleotides exist in solution as triple helices under the conditions of crystallization

A novel DNA duplex. A parallel-stranded DNA helix with Hoogsteen base pairing

We show here for the first time that a stable parallel double helix with Hoogsteen pairing can exist independently of the triple helix of which it is a component part. The experiments employ DNA oligonucleotides with mixed sequences of normal bases. These duplexes are distinct from previously reported ribopolynucleotide helices containing bulky substituents which prevent Watson-Crick pairing as well as from parallel duplexes with Donohue, or reversed Watson-Crick, pairing. Stoichiometry is established by mixing curves and gel electrophoresis. Tm depends linearly upon pH, increasing with acidity because of the need to protonate N3 of C. The Tm of the 20-mer studied here is 52° C at pH 5.2 and 0.1 M NaCl. At pH above 6, the molecule rearranges to form an antiparallel duplex with imperfect Watson-Crick pairing and loops, and the Tm is then independent of pH. The CD spectrum of the parallel duplex is very similar to that of the corresponding triple helix but quite different from that of the Watson-Crick helix. The infrared spectrum in the double bond region closely resembles that of the triple helix but, as with the CD, is quite different from that of the Watson-Crick duplex. The infrared spectra of the duplex and triple helix are also nearly identical in the region form 800 to 1000 cm-1, which is sensitive to backbone conformation. The only symmetry element present is a pseudorotational axis coincident with the helix axis of the parallel duplex as well as with the axis of the corresponding triple helix

Fibre-type X-ray diffraction patterns from single crystals of triple helical DNA

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