Emmanuel O. Balogun

Structure of the trypanosome cyanide-insensitive alternative oxidase

In addition to haem copper oxidases, all higher plants, some algae, yeasts, molds, metazoans, and pathogenic microorganisms such as Trypanosoma brucei contain an additional terminal oxidase, the cyanide-insensitive alternative oxidase (AOX). AOX is a diiron carboxylate protein that catalyzes the four-electron reduction of dioxygen to water by ubiquinol. In T. brucei, a parasite that causes human African sleeping sickness, AOX plays a critical role in the survival of the parasite in its bloodstream form. Because AOX is absent from mammals, this protein represents a unique and promising therapeutic target. Despite its bioenergetic and medical importance, however, structural features of any AOX are yet to be elucidated. Here we report crystal structures of the trypanosomal alternative oxidase in the absence and presence of ascofuranone derivatives. All structures reveal that the oxidase is a homodimer with the nonhaem diiron carboxylate active site buried within a four-helix bundle. Unusually, the active site is ligated solely by four glutamate residues in its oxidized inhibitor-free state; however, inhibitor binding induces the ligation of a histidine residue. A highly conserved Tyr220 is within 4 Å of the active site and is critical for catalytic activity. All structures also reveal that there are two hydrophobic cavities per monomer. Both inhibitors bind to one cavity within 4 Å and 5 Å of the active site and Tyr220, respectively. A second cavity interacts with the inhibitor-binding cavity at the diiron center. We suggest that both cavities bind ubiquinol and along with Tyr220 are required for the catalytic cycle for O2 reduction.

Quantitative changes in gastric mucosal glycoproteins: effect of cholinergic agonist and vagal nerve stimulation in the rat

The role of the vagus nerve and cholinergic mechanisms in the control of the rat gastric mucin and protein (PROT) release in vivo was investigated. Under urethane anaesthesia (1.25u2003gu2003kg−1), the rats had their gastric lumen perfused with saline. Mucus secretion was measured as a function of adherent mucus on the mucosa surface and the luminal content of sialic acids (SIA), galactose (GAL), pyruvate and PROT. Electrical stimulation of the vagi significantly increased the levels of mucus (3.23u2003±u2003025u2003μgu2003g−1 tissue, Pu2003<u20030.05), free sialic acid (FS) (0.18u2003±u20030.04u2003mgu2003mL−1, Pu2003<u20030.05) and PROT (0.25u2003±u20030.003u2003mgu2003mL−1, Pu2003<u20030.05) when compared with control animals. Bilateral cervical vagotomy had no significant effect on adherent mucus or basal levels of PROT, SIA and GAL (Pu2003>u20030.05) with respect to the control. In both vagotomized and vagal intact animals, the cholinergic agonist (carbachol, 200u2003mgu2003kg−1) significantly increased PROT, adherent mucus and FS (Pu2003<u20030.05) and decreased bound sialic acid (Pu2003>u20030.05). There were no visible haemorrhagic streaks on the gastric mucosa of vagotomized, vagal intact and carbachol‐treated animals. The results suggest that vagus nerve does not exert a tonic control on gastric glycoprotein secretion in vivo and that cholinergic effect on the mucus secreting cells may be implemented via the intrinsic nerves of the enteric nervous system.

Identification and Characterization of Sialidase-Like Activity in the Developmental Stages of Amblyomma variegatum

ABSTRACT Amblyomma variegatum F. are obligate hematophagous ectoparasites of livestock that serve as the vectors of Ehrlichia ruminantium (formerly known as Cowdria ruminantium), the causative agent of heartwater disease. In the light of the fact that they are blood-feeding, their salivary glands play prominent role in their acquisition of nutrients from the bloodmeal. Sialic acids are a major component of glycoprotein in mammalian blood fluid and cells. Sialome of hard ticks is still sparse. Here, for the first time, the possible expression of sialidase in A. variegatum was investigated. Our finding established the presence of type II sialidase-like activity in the three stages (larva, nymph, and adult) of the fed and unfed tick. There was no statistically significant difference in sialidase activity in the various stages of this ectoparasite (P > 0.05). The enzyme was purified by combination of salting out and ion exchange chromatography on DEAE—cellulose and hydroxylapatite columns. Characterization of the enzyme revealed that it is optimally active at 40°C and pH 5.5, and is activated by bivalent cations Zn2+ or Fe2+. The enzyme has a Km of 0.023 mM and Vmax of 0.16 millimol/min with Fetuin as the substrate. To assess the susceptibility of some mammalian cells to the tick sialidase, we prepared erythrocyte ghost cells from different animals, which were incubated with the enzyme. Results revealed that the ruminant cells were better substrates. Our work and findings contribute to the preliminary characterization of the A. variegatum salivary proteome, and may pave way to the development of new acaricides.

SAR of 4-Alkoxybenzoic Acid Inhibitors of the Trypanosome Alternative Oxidase

The SAR of 4-hydroxybenzaldehyde inhibitors of the trypanosome alternative oxidase (TAO), a critical enzyme for the respiration of bloodstream forms of trypanosomes, was investigated. Replacing the aldehyde group with a methyl ester resulted in a 10-fold increase in TAO inhibition and activity against T. brucei. Remarkably, two analogues containing the 2-hydroxy-6-methyl scaffold (9e and 16e) displayed single digit nanomolar TAO inhibition, which constitute the most potent 4-alkoxybenzoic acid derivatives described to date. 9e was 50-times more potent against TAO and 10-times more active against T. brucei compared to its benzaldehyde analogue 1. The farnesyl derivative 16e was as potent a TAO inhibitor as ascofuranone with IC50 = 3.1 nM. Similar to ascofuranone derivatives, the 2-hydroxy and 6-methyl groups seemed essential for low nanomolar TAO inhibition of acid derivatives, suggesting analogous binding interactions with the TAO active site.

Pro-inflammatory Cytokine Response and Genetic Diversity in Merozoite Surface Protein 2 of Plasmodium falciparum Isolates from Nigeria

Background: Polymorphisms in Plasmodium falciparum merozoite surface protein-2 (msp-2) and associated parasite genetic diversity which varies between malaria-endemic regions remain a limitation in malaria vaccine development. Pro-inflammatory cytokines are important in immunity against malaria, understanding the influence of genetic diversity on cytokine response is important for effective vaccine design. Methods: P. falciparum isolates obtained from 300 Nigerians with uncomplicated falciparum malaria at Ijede General Hospital, Ijede (IJE), General Hospital Ajeromi, Ajeromi (AJE) and Saint Kizito Mission Hospital, Lekki, were genotyped by nested polymerase chain reaction of msp-2 block 3 while ELISA was used to determine the pro-inflammatory cytokine response to describe the genetic diversity of P. falciparum. Results: Eighteen alleles were observed for msp-2 loci. Of the 195 isolates, 61 (31.0%) had only FC27-type alleles, 38 (19.7%) had only 3D7-type alleles, and 49.3% had multiple parasite lines with both alleles. Band sizes were 275–625 bp for FC27 and 150–425 bp for 3D7. Four alleles were observed from LEK, 2 (375–425 bp) and 2 (275–325 bp) of FC27-and 3D7-types, respectively; 12 alleles from AJE, 9 (275–625 bp) and 3 (325–425 bp) of FC27-types and 3D7-types, respectively; while IJE had a total of 12 alleles, 9 (275–625 bp) and 3 (325–425 bp) of FC27-types and 3D7-types, respectively. Mean multiplicity of infection (MOI) was 1.54. Heterozygosity (HE) ranged from 0.77 to 0.87 and was highest for IJE (0.87). Cytokine response was higher among <5 years and was significantly associated with MOI (P > 0.05) but with neither parasite density nor infection type. Conclusion: P. falciparum genetic diversity is extensive in Nigeria, protection via pro-inflammatory cytokines have little or no interplay with infection multiplicity.