Properties of phosphoenolpyruvate mutase, the first enzyme in the aminoethylphosphonate biosynthetic pathway in Trypanosoma cruzi.

Phosphoenolpyruvate (PEP) mutase catalyzes the conversion of phosphoenolpyruvate to phosphonopyruvate, the initial step in the formation of many naturally occurring phosphonate compounds. The phosphonate compound 2-aminoethylphosphonate is present as a component of complex carbohydrates on the surface membrane of many trypanosomatids including glycosylinositolphospholipids of Trypanosoma cruzi. Using partial sequence information from the T.

Evidence for DNA translocation by the ISWI chromatin-remodeling enzyme.

The ISWI proteins form the catalytic core of a subset of ATP-dependent chromatin-remodeling activities. Here, we studied the interaction of the ISWI protein with nucleosomal substrates. We found that the ability of nucleic acids to bind and stimulate the ATPase activity of ISWI depends on length.

Crystal structure of activated ModE reveals conformational changes involving both oxyanion and DNA-binding domains.

ModE is a bacterial transcriptional regulator that orchestrates many aspects of molybdenum metabolism by binding to specific DNA sequences in a molybdate-dependent fashion. We present the crystal structure of Escherichia coli ModE in complex with molybdate, which was determined at 2.75A from a merohedrally twinned crystal (twin fraction approximately 0.

Siglecs: sialic-acid-binding immunoglobulin-like lectins in cell-cell interactions and signalling.

Siglecs are sialic-acid-binding immunoglobulin-like lectins involved in cell-cell interactions and signalling functions in the haemopoietic, immune and nervous systems. Significant advances have been made in our understanding of the link between carbohydrate recognition and signalling for two well-characterised siglecs, CD22 and myelin-associated glycoprotein. Over the past few years, several novel siglecs have been discovered through genomics and functional screens.

Regulation of Tbx3 expression by anteroposterior signalling in vertebrate limb development.

Tbx3, a T-box gene family member related to the Drosophila gene optomotor blind (omb) and encoding a transcription factor, is expressed in anterior and posterior stripes in developing chick limb buds. Tbx3 haploinsufficiency has been linked with the human condition ulnar-mammary syndrome, in which predominantly posterior defects occur in the upper limb. Omb is expressed in Drosophila wing development in response to a signalling cascade involving Hedgehog and Dpp.

Partial structure of glutamic acid and alanine-rich protein, a major surface glycoprotein of the insect stages of Trypanosoma congolense.

The tsetse fly transmitted salivarian trypanosome, Trypanosoma congolense of the subgenus Nanomonas, is the most significant of the trypanosomes with respect to the pathology of livestock in sub-Saharan Africa. Unlike the related trypanosome Trypanosoma brucei of the subgenus Trypanozoon, the major surface molecules of the insect stages of T. congolense are poorly characterized.

Cloning of Trypanosoma brucei and Leishmania major genes encoding the GlcNAc-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol biosynthesis that is essential to the African sleeping sickness parasite.

The second step of glycosylphosphatidylinositol anchor biosynthesis in all eukaryotes is the conversion of D-GlcNAcalpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-diacylglycerol (GlcNAc-PI) to d-GlcNalpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-diacylglycerol by GlcNAc-PI de-N-acetylase. The genes encoding this activity are PIG-L and GPI12 in mammals and yeast, respectively. Fragments of putative GlcNAc-PI de-N-acetylase genes from Trypanosoma brucei and Leishmania major were identified in the respective genome project data bases.

The glycoforms of a Trypanosoma brucei variant surface glycoprotein and molecular modeling of a glycosylated surface coat.

The plasma membrane of the African sleeping sickness parasite Trypanosoma brucei is covered with a dense, protective surface coat. This surface coat is a monolayer of five million variant surface glycoprotein (VSG) dimers that form a macromolecular diffusion barrier. The surface coat protects the parasite from the innate immune system and, through antigenic variation, the specific host immune response.

A single enzyme catalyses formation of Trypanothione from glutathione and spermidine in Trypanosoma cruzi.

Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (l-gamma-glutamyl-cysteinyl-glycine) and spermidine to form trypanothione [N(1),N(8)-bis(glutathionyl)spermidine], a metabolite involved in defense against chemical and oxidant stress and other biosynthetic functions. In Crithidia fasciculata, trypanothione is synthesized from GSH and spermidine via the intermediate glutathionylspermidine in two distinct ATP-dependent reactions catalyzed by glutathionylspermidine synthetase (GspS; EC ) and trypanothione synthetase (TryS; EC ), respectively. Here we have cloned a single copy gene (TcTryS) from Trypanosoma cruzi encoding a protein with 61% sequence identity with CfTryS but only 31% with CfGspS.

Simultaneous quantification of cell motility and protein-membrane-association using active contours.

We present a new method for the quantification of dynamic changes in fluorescence intensities at the cell membrane of moving cells. It is based on an active contour method for cell-edge detection, which allows tracking of changes in cell shape and position. Fluorescence intensities at specific cortical subregions can be followed in space and time and correlated with cell motility.

Galactose metabolism is essential for the African sleeping sickness parasite Trypanosoma brucei.

The tsetse fly-transmitted protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and the cattle disease Nagana. The bloodstream form of the parasite uses a dense cell-surface coat of variant surface glycoprotein to escape the innate and adaptive immune responses of the mammalian host and a highly glycosylated transferrin receptor to take up host transferrin, an essential growth factor. These glycoproteins, as well as other flagellar pocket, endosomal, and lysosomal glycoproteins, are known to contain galactose.

A new expression vector for Crithidia fasciculata and Leishmania.

Crithidia fasciculata is a monogenetic parasite of insects. It grows in fully defined media without requiring serum, which facilitates biochemical analysis. We have constructed a series of expression systems that allows expression of transfected genes in the kinetoplastid protozoa Crithidia and Leishmania.

Metabolic pathway analysis in trypanosomes and malaria parasites.

Identification of novel drug targets is required for the development of new classes of drugs to overcome drug resistance and replace less efficacious treatments. In theory, knowledge of the entire genome of a pathogen identifies every potential drug target in any given microbe. In practice, the sheer complexity and the inadequate or inaccurate annotation of genomic information makes target identification and selection somewhat more difficult.

Passive acquisition of ligand by the MopII molbindin from Clostridium pasteurianum: structures of apo and oxyanion-bound forms.

MopII from Clostridium pasteurianum is a molbindin family member. These proteins may serve as intracellular storage facilities for molybdate, which they bind with high specificity. High resolution structures of MopII in a number of states, including the first structure of an apo-molbindin, together with calorimetric data, allow us to describe ligand binding and provide support for the proposed storage function of the protein.

Crystallization and X-ray diffraction measurements on recombinant molbindin, MopII, from Clostridium pasteurianum.

Clostridium pasteurianum carries three genes termed mopI, II and III encoding three molbindin isoforms, one of which has been cloned, the gene product expressed in high yield and crystallized using the hanging-drop vapour-diffusion method. Well ordered monoclinic crystals in two different crystal forms, both with space group C2, were obtained in the presence and absence of Na(2)MoO(4) and Na(2)WO(4). Ligand-bound MopII crystallized with polyethylene glycol (PEG) 400 as a precipitant, whereas apo MopII required PEG 6000.

Purification, cloning and characterization of a GPI inositol deacylase from Trypanosoma brucei.

Inositol acylation is an obligatory step in glycosylphosphatidylinositol (GPI) biosynthesis whereas mature GPI anchors often lack this modification. The GPI anchors of Trypanosoma brucei variant surface glycoproteins (VSGs) undergo rounds of inositol acylation and deacylation during GPI biosynthesis and the deacylation reactions are inhibited by diisopropylfluorophosphate (DFP). Inositol deacylase was affinity labelled with [3H]DFP and purified.

Molecular characterisation of mitochondrial and cytosolic trypanothione-dependent tryparedoxin peroxidases in Trypanosoma brucei.

In trypanosomatids, removal of hydrogen peroxide and other aryl and alkyl peroxides is achieved by the NADPH-dependent trypanothione peroxidase system, whose components are trypanothione reductase (TRYR), trypanothione, tryparedoxin (TRYX) and tryparedoxin peroxidase (TRYP). Here, we report the cloning of a multi-copy tryparedoxin peroxidase gene (TRYP1) from Trypanosoma brucei brucei encoding a protein with two catalytic VCP motifs similar to the cytosolic TRYP from Crithidia fasciculata. In addition, we characterise a novel single copy gene encoding a second tryparedoxin peroxidase (TRYP2).

Difference in XTcf-3 dependency accounts for change in response to beta-catenin-mediated Wnt signalling in Xenopus blastula.

Wnt signalling functions in many tissues and during different stages of animal development to produce very specific responses. In early Xenopus embryos there is a dramatic change in response to Wnt signalling within only a few hours of development. Wnt signalling in very early embryos leads to a dorsalising response, which establishes the endogenous dorsal axis.

Crystallization and preliminary X-ray diffraction studies of recombinant Escherichia coli 4-diphosphocytidyl-2-C-methyl-D-erythritol synthetase.

Diphosphocytidyl-methylerythritol (DPCME) synthetase is involved in the mevalonate-independent pathway of isoprenoid biosynthesis, where it catalyses the formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol from 2-C-methyl-D-erythritol 4-phosphate and CTP. The Escherichia coli enzyme has been cloned, expressed in high yield, purified and crystallized. Elongated tetragonal prismatic crystals grown by the hanging-drop vapour-diffusion method using polyethylene glycol (PEG) 4000 as the precipitant belong to space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = b = 73.

ATP-dependent chromatin remodeling activities.

Genetic and biochemical approaches have indicated that the packaging of DNA into chromatin can be repressive to transcription. ATP-dependent chromatin remodelling activities can facilitate transcription from chromatin templates. Consistent with this, biochemical assays have shown that the action of ATP-dependent chromatin remodelling activities increase the accessibility of DNA within chromatin templates.

Siglecs, sialic acids and innate immunity.

Siglecs are members of the Ig superfamily that bind to sialic acid (Sia) and are mainly expressed by cells of the hematopoietic system. Until three years ago, only four Siglecs were known, namely sialoadhesin, CD22, myelin-associated glycoprotein and CD33. Since then, a further six human CD33-related Siglecs with features of inhibitory receptors have been identified and shown to be expressed by discrete subsets of leukocytes.

Pteridine reductase mechanism correlates pterin metabolism with drug resistance in trypanosomatid parasites.

Pteridine reductase (PTR1) is a short-chain reductase (SDR) responsible for the salvage of pterins in parasitic trypanosomatids. PTR1 catalyzes the NADPH-dependent two-step reduction of oxidized pterins to the active tetrahydro-forms and reduces susceptibility to antifolates by alleviating dihydrofolate reductase (DHFR) inhibition. Crystal structures of PTR1 complexed with cofactor and 7,8-dihydrobiopterin (DHB) or methotrexate (MTX) delineate the enzyme mechanism, broad spectrum of activity and inhibition by substrate or an antifolate.

Structure of the macrocycle thiostrepton solved using the anomalous dispersion contribution of sulfur.

The structure of a tetragonal crystal form of thiostrepton has been solved using the anomalous dispersive effects of five S atoms from high-redundancy data collected to 1.33 A resolution at the Cu Kalpha wavelength. Data measured to 1.

Identification, characterization and leucocyte expression of Siglec-10, a novel human sialic acid-binding receptor.

Here we characterize Siglec-10 as a new member of the Siglec family of sialic acid-binding Ig-like lectins. A full-length cDNA was isolated from a human spleen library and the corresponding gene identified. Siglec-10 is predicted to contain five extracellular Ig-like domains and a cytoplasmic tail containing three putative tyrosine-based signalling motifs.