Allosteric regulation accompanied by oligomeric state changes of Trypanosoma brucei GMP reductase through cystathionine-ß-synthase domain.

Guanosine 5'-monophosphate reductase (GMPR) is involved in the purine salvage pathway and is conserved throughout evolution. Nonetheless, the GMPR of Trypanosoma brucei (TbGMPR) includes a unique structure known as the cystathionine-ß-synthase (CBS) domain, though the role of this domain is not fully understood. Here, we show that guanine and adenine nucleotides exert positive and negative effects, respectively, on TbGMPR activity by binding allosterically to the CBS domain. The present structural analyses revealed that TbGMPR forms an octamer that shows a transition between relaxed and twisted conformations in the absence and presence of guanine nucleotides, respectively, whereas the TbGMPR octamer dissociates into two tetramers when ATP is available instead of guanine nucleotides. These findings demonstrate that the CBS domain plays a key role in the allosteric regulation of TbGMPR by facilitating the transition of its oligomeric state depending on ligand nucleotide availability.

Novel Characteristics of Trypanosoma brucei Guanosine 5'-monophosphate Reductase Distinct from Host Animals.

The metabolic pathway of purine nucleotides in parasitic protozoa is a potent drug target for treatment of parasitemia. Guanosine 5'-monophosphate reductase (GMPR), which catalyzes the deamination of guanosine 5'-monophosphate (GMP) to inosine 5'-monophosphate (IMP), plays an important role in the interconversion of purine nucleotides to maintain the intracellular balance of their concentration. However, only a few studies on protozoan GMPR have been reported at present. Herein, we identified the GMPR in Trypanosoma brucei, a causative protozoan parasite of African trypanosomiasis, and found that the GMPR proteins were consistently localized to glycosomes in T. brucei bloodstream forms. We characterized its recombinant protein to investigate the enzymatic differences between GMPRs of T. brucei and its host animals. T. brucei GMPR was distinct in having an insertion of a tandem repeat of the cystathionine ß-synthase (CBS) domain, which was absent in mammalian and bacterial GMPRs. The recombinant protein of T. brucei GMPR catalyzed the conversion of GMP to IMP in the presence of NADPH, and showed apparent affinities for both GMP and NADPH different from those of its mammalian counterparts. Interestingly, the addition of monovalent cations such as K+ and NH4+ to the enzymatic reaction increased the GMPR activity of T. brucei, whereas none of the mammalian GMPR's was affected by these cations. The monophosphate form of the purine nucleoside analog ribavirin inhibited T. brucei GMPR activity, though mammalian GMPRs showed no or only a little inhibition by it. These results suggest that the mechanism of the GMPR reaction in T. brucei is distinct from that in the host organisms. Finally, we demonstrated the inhibitory effect of ribavirin on the proliferation of trypanosomes in a dose-dependent manner, suggesting the availability of ribavirin to develop a new therapeutic agent against African trypanosomiasis.

Late stage infection in sleeping sickness.

At the turn of the 19(th) century, trypanosomes were identified as the causative agent of sleeping sickness and their presence within the cerebrospinal fluid of late stage sleeping sickness patients was described. However, no definitive proof of how the parasites reach the brain has been presented so far. Analyzing electron micrographs prepared from rodent brains more than 20 days after infection, we present here conclusive evidence that the parasites first enter the brain via the choroid plexus from where they penetrate the epithelial cell layer to reach the ventricular system. Adversely, no trypanosomes were observed within the parenchyma outside blood vessels. We also show that brain infection depends on the formation of long slender trypanosomes and that the cerebrospinal fluid as well as the stroma of the choroid plexus is a hostile environment for the survival of trypanosomes, which enter the pial space including the Virchow-Robin space via the subarachnoid space to escape degradation. Our data suggest that trypanosomes do not intend to colonize the brain but reside near or within the glia limitans, from where they can re-populate blood vessels and disrupt the sleep wake cycles.

New anti-malarial phenylpropanoid conjugated iridoids from Morinda morindoides.

A new phenylpropanoid conjugated iridoid together with four known congeners was isolated from Morinda morindoides, used for the therapy of malaria traditionally in some African countries, as anti-malarial principles through bioassay-guided separation. Furthermore, their absolute stereostructures were unambiguously established by a combination of modified Mosher's method and chemical correlation.

Molecular basis for prostaglandin production in hosts and parasites.

Prostaglandins (PGs) comprise a family of structurally related bioactive lipid mediators that are involved in various symptoms associated with parasitic diseases. The molecular mechanisms of PG biosynthesis in animals have been studied extensively. Currently, several lines of evidence link their production with parasites. In this review we discuss the roles of PGs in parasite pathogenesis and physiology and the recent advances in our understanding of the enzymology of PG production in various parasites.

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania.

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania.

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.

Crystallization and preliminary X-ray crystallographic studies of Trypanosoma brucei prostaglandin F(2 alpha) synthase.

Prostaglandin F(2 alpha) is a potent mediator of various physiological and pathological processes. Trypanosoma brucei prostaglandin F(2 alpha) synthase (TbPGFS) catalyzes the NADPH-dependent reduction of 9,11-endoperoxide PGH(2) to PGF(2 alpha), and could thus be involved in the elevation of the PGF(2 alpha) concentration during African trypanosomiasis. In the present report, the purification and crystallization of recombinant TbPGFS are described. The active recombinant enzyme was crystallized by the hanging-drop vapor-diffusion meth-od using ammonium sulfate as a precipitant. The crystal belonged to a tetragonal space group, P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters of a = b = 112.3 A, and c = 140.0 A. Native data up to 2.6 A resolution were collected from the crystal using our home facility.

A key role for old yellow enzyme in the metabolism of drugs by Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas' disease. So far, first choice anti-chagasic drugs in use have been shown to have undesirable side effects in addition to the emergence of parasite resistance and the lack of prospect for vaccine against T. cruzi infection. Thus, the isolation and characterization of molecules essential in parasite metabolism of the anti-chagasic drugs are fundamental for the development of new strategies for rational drug design and/or the improvement of the current chemotherapy. While searching for a prostaglandin (PG) F(2alpha) synthase homologue, we have identified a novel "old yellow enzyme" from T. cruzi (TcOYE), cloned its cDNA, and overexpressed the recombinant enzyme. Here, we show that TcOYE reduced 9,11-endoperoxide PGH(2) to PGF(2alpha) as well as a variety of trypanocidal drugs. By electron spin resonance experiments, we found that TcOYE specifically catalyzed one-electron reduction of menadione and beta-lapachone to semiquinone-free radicals with concomitant generation of superoxide radical anions, while catalyzing solely the two-electron reduction of nifurtimox and 4-nitroquinoline-N-oxide drugs without free radical production. Interestingly, immunoprecipitation experiments revealed that anti-TcOYE polyclonal antibody abolished major reductase activities of the lysates toward these drugs, identifying TcOYE as a key drug-metabolizing enzyme by which quinone drugs have their mechanism of action.

Immunohistochemical localization of microsomal PGE synthase-1 and cyclooxygenases in male mouse reproductive organs.

We investigated the tissue distribution and cellular localization of microsomal PGE synthase-1 (mPGES-1) and cyclooxygenase (COX)-1 and -2 in the male mouse reproductive organs. Northern blotting revealed that the mPGES-1 mRNA was expressed intensely in the epididymis and weakly in the lung, spleen, skin, kidney, colon, and brain. In the male reproductive tract, the expression of mPGES-1 increased from the testis to the cauda epididymis and was highest in the vas deferens when examined by Northern blotting, RT-PCR, and Western blotting. By immunohistochemistry, mPGES-1 was detected in Leydig cells of the testis and in epithelial cells of the epididymis, vas deferens, and seminal vesicles. In addition, the caput and cauda regions of the epididymis and the vas deferens in this order showed a progressive increase in the expression of COX-1 mRNA and immunoreactivity, whereas COX-2 was dominantly expressed in the vas deferens. COX-1 was localized in epithelial cells of the caput, corpus and cauda epididymis and of the vas deferens, and COX-2 was evident in epithelial cells of the distal cauda epididymis and vas deferens. These results show that mPGES-1 is expressed coordinately with COX-1 and COX-2 and is involved in PGE(2) production in male genital organs.

Biochemical characterization of mouse microsomal prostaglandin E synthase-1 and its colocalization with cyclooxygenase-2 in peritoneal macrophages.

We cloned the cDNA for mouse microsomal prostaglandin (PG) E synthase-1 (mPGES-1) and expressed the recombinant enzyme in Escherichia coli. The membrane fraction containing recombinant mPGES-1 catalyzed the isomerization of PGH2 to PGE2 in the presence of GSH with K(m) values of 130 microM for PGH2 and 37 microM for GSH, a turnover number of 600 min(-1), and a k(cat)/K(m) ratio of 4.6 min(-1) microM(-1). Recombinant mPGES-1 was purified and used to generate a polyclonal antibody highly specific for mPGES-1. The antibody showed a single band on Western blotting of microsomal fractions from lipopolysaccharide-treated mouse peritoneal macrophages. Northern and Western blotting analyses revealed that mPGES-1 was induced together with cyclooxygenase-2 in mouse macrophages after treatment of the cells with lipopolysaccharide. Confocal immunofluorescence microscopy revealed that both mPGES-1 and cyclooxygenase-2 were colocalized in the lipopolysaccharide-treated macrophages. Taken together, these results demonstrate that mPGES-1 is an efficient downstream enzyme for the production of PGE2 in the activated macrophages treated by lipopolysaccharide.