Biomedical subjects

Y Yuthavong

Publications and source records attributed to Y Yuthavong.

At least 37 records · Page 2Linked to original sources

PubMed1995

Antimalarial sesquiterpenes from tubers of Cyperus rotundus: structure of 10,12-peroxycalamenene, a sesquiterpene endoperoxide.

Activity-guided investigation of Cyperus rotundus tubers led to the isolation of patchoulenone, caryophyllene alpha-oxide, 10,12-peroxycalamenene and 4,7-dimethyl-1-tetralone. The antimalarial activities of these compounds are in the range of EC50 10(-4)-10(-6) M, with the novel endoperoxide sesquiterpene, 10,12-peroxycalamenene, exhibiting the strongest effect at EC50 2.33 x 10(-6) M.

Animals↗

PubMed1994

Resistance to artemisinin of malaria parasites (Plasmodium falciparum) infecting alpha-thalassemic erythrocytes in vitro. Competition in drug accumulation with uninfected erythrocytes.

Plasmodium falciparum infecting hemoglobin (Hb)H and/or Hb Constant Spring erythrocytes has higher resistance to artemisinin in vitro than when infecting normal erythrocytes. This is due to low drug accumulation of infected erythrocytes resulting from competition with uninfected variant erythrocytes, which have a higher accumulation capacity than genetically normal cells. Drug accumulation of the parasite was shown to be saturable and dependent on metabolic energy. The 50% inhibitory concentrations (IC50's) for the parasite in HbH/Hb Constant Spring erythrocytes were decreased when normal erythrocytes were added to the infected cells, and correspondingly, the IC50's in normal erythrocytes were increased when HbH/Hb Constant Spring erythrocytes were added to the infected cells. The changes of IC50 corresponded to the variation in drug accumulation of mixtures of normal and variant erythrocytes of different compositions. The IC50's for the parasite in variant erythrocytes were also greatly decreased when the hematocrit of the culture was lowered, while the IC50's in normal erythrocytes were independent of the hematocrit. The increase in IC50 values for the parasites infecting variant erythrocytes was also related to the decrease in parasite accumulation, indicating that drug accumulation capacity of the parasite also has a role in determining drug sensitivity. Artemisinin sensitivity therefore is determined by its accessibility to the parasite, which is decreased in infected variant erythrocytes.

Animals↗

PubMed1993

The dihydrofolate reductase domain of Plasmodium falciparum thymidylate synthase-dihydrofolate reductase. Gene synthesis, expression, and anti-folate-resistant mutants.

A 693-base pair gene coding for the 27,132-dalton dihydrofolate reductase (DHFR) domain of the thymidylate synthase-dihydrofolate reductase (TS-DHFR) bifunctional protein of Plasmodium falciparum was designed to have Escherichia coli codon preference and multiple unique restriction sites and was chemically synthesized. The gene was overexpressed (> 50% total cellular protein) in E. coli as insoluble inclusion bodies which could be unfolded and refolded to recover soluble enzyme activity. The refolded DHFR was purified by methotrexate-Sepharose affinity chromatography to give the homogeneous enzyme. Active site titration with methotrexate revealed that the purified protein was fully active. The purified DHFR migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent mass of approximately 30 kDa, and gel filtration showed that the protein is a monomer. The yield of purified enzyme was about 5-6 mg/liter of bacterial culture. Kinetic properties of the purified recombinant DHFR were similar to those reported for wild type bifunctional TS-DHFR. Cassette mutagenesis of the synthetic gene was performed to give the S108N and the N51I + S108N mutants which provided DHFRs analogous to pyrimethamine-resistant mutants found in nature.

Amino Acid Sequence↗

PubMed1993

Protection against malaria by thalassaemia and haemoglobin variants.

Recent epidemiological evidence has given increasing support to Haldane's 1949 hypothesis that heterozygotes for such genetic disorders as thalassaemia might be protected against malaria, hence explaining the high gene frequencies for such disorders in endemic areas. As discussed here by Yongyuth Yuthavong and Prapon Wilairat, the possible cellular mechanisms, although still unclear, are emerging from in vitro studies which increasingly point to the importance of immune clearance mechanisms in some cases (such as alpha-thalassaemia and haemoglobin E). In other situations, decreased survival of the intra-erythrocytic parasite or decreased parasite invasion of the variant red blood cells may explain the protective effect. Whatever the cellular mechanisms are, the ultimate decisive factor is the relative fitness of the infected variant host, which may not be simply extrapolated from the cellular studies.

Journal Article↗

PubMed1993

The methionine synthesis cycle and salvage of methyltetrahydrofolate from host red cells in the malaria parasite (Plasmodium falciparum).

Plasmodium falciparum, P. knowlesi and P. chabaudi showed a significant activity of methylenetetrahydrofolate reductase (MTHFR). The presence of this enzyme completes the methionine synthesis cycle, in which the one-carbon fragment from serine side-chain can be transferred to methionine. However, while metabolic labelling of methionine from L-3 [14C]serine could not be demonstrated in P. falciparum, the significance of MTHFR was implicated by a novel pathway for salvage of exogenous 5-methyltetrahydrofolate from the host cell. The methyl group of the cofactor was incorporated into methionine, and the folate cofactor was found in the same pool as that derived from de novo synthesis with p-aminobenzoic acid as the precursor, shown previously as polyglutamylated 5-methyltetrahydrofolate. It is proposed from these results that the function of MTHFR and the methionine synthesis cycle is not the supply of methionine, but the generation of active folate cofactors from more stable precursors salvaged by the parasites.

Animals↗

PubMed1993

Iron-dependent free radical generation from the antimalarial agent artemisinin (qinghaosu).

Artemisinin is an important new antimalarial agent containing a bridged endoperoxide. The in vitro antimalarial activity of an artemisinin derivative, arteether, is antagonized by two iron chelators, pyridoxal benzoylhydrazone and 1,2-dimethyl-3-hydroxypyrid-4-one. Similarly, the acute toxicity of artemisinin in mice is antagonized by another chelator, deferoxamine-hydroxyethylstarch. A combination of artemisinin and hemin oxidizes erythrocyte membrane thiols in vitro, and this oxidation is also inhibited by an iron chelator. Thus, iron plays a role in the mechanisms of action and toxicity of artemisinin. The combination of artemisinin and hemin also decreases erythrocyte deformability. Iron probably catalyzes the generation of free radicals from artemisinin since alpha-tocopherol antagonizes the thiol-oxidizing activity of artemisinin and since a spin-trapped free radical signal can be seen by electron paramagnetic resonance only when artemisinin is incubated in the presence of iron.

Animals↗

PubMed1992

High-performance liquid chromatographic determination of dihydroorotate dehydrogenase of Plasmodium falciparum and effects of antimalarials on enzyme activity.

A reversed-phase high-performance liquid chromatographic technique for the determination of dihydroorotate dehydrogenase in Plasmodium falciparum was developed. The assay was applied to the evaluation of the effects of several antimalarial drugs on the enzyme. Treatment of both the asexual and gametocyte stages of P. falciparum in culture with menoctone, primaquine or the primaquine derivative WR 238605 led to depression of the enzyme activity, although the drugs did not appear to inhibit the enzyme directly.

Aminoquinolines↗

PubMed1992

Subunit complementation of thymidylate synthase.

Each of the two active sites of thymidylate synthase contains amino acid residues contributed by the other subunit. For example, Arg-178 of one monomer binds the phosphate group of the substrate dUMP in the active site of the other monomer [Hardy et al. (1987) Science 235, 448-455]. Inactive mutants of such residues should combine with subunits of other inactive mutants to form heterodimeric hybrids with one functional active site. In vivo and in vitro approaches were used to test this hypothesis. In vivo complementation was accomplished by cotransforming plasmid mixtures encoding pools of inactive Arg-178 mutants and pools of inactive Cys-198 mutants into a host strain deficient in thymidylate synthase. Individual inactive mutants of Arg-178 were also cotransformed with the C198A mutant. Subunit complementation was detected by selection or screening for transformants which grew in the absence of thymidine, and hence produced active enzyme. Many mutants at each position representing a wide variety of size and charge supported subunit complementation. In vitro complementation was accomplished by reversible dissociation and unfolding of mixtures of purified individual inactive Arg-178 and Cys-198 mutant proteins. With the R178F + C198A heterodimer, the Km values for dUMP and CH2H4folate were similar to those of the wild-type enzyme. By titrating C198A with R178F under unfolding-refolding conditions, we were able to calculate the kcat value for the active heterodimer. The catalytic efficiency of the single wild-type active site of the C198A + R178F heterodimer approaches that of the wild-type enzyme.

Amino Acid Sequence↗

PubMed1992

Reduced deformability of thalassemic erythrocytes and erythrocytes with abnormal hemoglobins and relation with susceptibility to Plasmodium falciparum invasion.

A number of genetically variant erythrocytes showed decreased deformability of both intact cells and membranes prepared therefrom as measured by laser diffractometry. Erythrocytes associated with minor or no clinical symptoms (eg, alpha-thalassemia traits, hemoglobin [Hb] E trait, Hb Constant Spring trait), which showed only a minimal decrease in deformability, were, in general, invaded efficiently by the malarial parasite Plasmodium falciparum. Other variant erythrocytes (beta-thalassemia/Hb E, homozygous Hb E, homozygous Hb Constant Spring, Hb H, Hb H/Hb Constant Spring) with low deformability showed different degrees of reduction in invasion susceptibility, most of which were less than proportional with deformability decrease. It is concluded that parasite invasion is only weakly related to gross cell deformability, which in turn depends on various factors other than membrane deformability.

Erythrocyte Deformability↗

PubMed1990

Heterologous expression of active thymidylate synthase-dihydrofolate reductase from Plasmodium falciparum.

The coding sequence of the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from a moderately pyrimethamine-resistant strain (HB3) of Plasmodium falciparum was assembled in a pUC expression vector. The coding sequence possesses unique Nco1 and Xba1 sites which flank 243 bp of the DHFR gene that include all point mutations thus far linked to pyrimethamine resistance. Wild-type (3D7) and highly pyrimethamine-resistant (7G8) TS-DHFRs were made from this vector by cassette mutagenesis using Nco1-Xba1 fragments from the corresponding cloned TS-DHFR genes. Catalytically active recombinant TS-DHFRs were expressed in Escherichia coli, albeit at low levels. Both TS and DHFR coeluted upon gel filtration and copurified upon affinity and anion exchange chromatography. Gel filtration and SDS-PAGE indicated that the enzyme was a dimer with identical 67-kDa subunits, characteristic of protozoan TS-DHFRs. Amino-terminal sequencing gave 10 amino acids which perfectly matched the sequence predicted from the nucleotide sequence. The recombinant TS-DHFR was purified to homogeneity by 10-formylfolate affinity chromatography followed by Mono Q FPLC. The inhibition properties of pyrimethamine toward the purified recombinant enzymes show that the point mutations are the molecular basis of pyrimethamine resistance in P. falciparum.

Amino Acid Sequence↗

PubMed1990

Folate and cobalamin metabolism in Plasmodium falciparum.

During the past several years, Jerapan Krungkrai, H. Kyle Webster and Yongyuth Yuthavong have characterized the metabolic pathway of folate biosynthesis and folate-dependent reactions, including the cobalamin-dependent activity of methionine synthase, in P. falciparum grown in vitro. In this review, they discuss the implications of this work for understanding the mechanism of pyrimethamine resistance and the importance of cloning the dihydrofolate reductase gene. In addition, the role of cobalamin in P. falciparum will be considered. Interference with cobalamin use may represent a new target for combating the parasite.

Journal Article↗

PubMed1990

Increased susceptibility of malaria-infected variant erythrocytes to the mononuclear phagocyte system.

The interactions of the mononuclear phagocyte system with Plasmodium falciparum-infected genetically variant erythrocytes may result in a significant protection for the host. Infected hemoglobin (Hb) EE and Hb EA erythrocytes are more susceptible to phagocytosis by monocytes than are infected Hb AA erythrocytes. The increased susceptibility to phagocytosis of infected erythrocytes was also found for a number of genetic variants involving the alpha-globin chain, namely, alpha-thal 1 trait (--/alpha alpha), alpha-thal 2 trait (-alpha/alpha alpha), Hb H (--/-alpha), Hb H/Hb Constant Spring (CS) (--/alpha CS alpha), Hb CS trait, and homozygous Hb CS erythrocytes. In addition, oxidative damage from hydrogen peroxide, produced in simulation of macrophages, led to much more effective killing of parasites in glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes than in normal ones. Parasites infecting Hb H/Hb CS also showed an enhanced sensitivity to hydrogen peroxide.

Animals↗

PubMed1989

Mitochondria as the site of action of tetracycline on Plasmodium falciparum.

Rhodamine 123 (Rh 123) was used as a fluorescent probe for the mitochondria of the malarial parasite Plasmodium falciparum. On treatment with tetracycline in vitro, a marked decrease in the percentage of parasites with Rh123 fluorescence in the mitochondria was observed in parallel with an increase in the percentage of parasites with abnormal morphology during onset of decrease in parasitemia. Similar results were obtained, over a shorter time period, with 2,4-dinitrophenol. However, the percentage of parasites with fluorescence did not decrease with increase in parasite abnormal morphology or decrease in parasitemia on treatment with pyrimethamine or cycloheximide. Isoelectric focusing-SDS gel electrophoresis of radiolabelled parasite proteins showed two components of 95 and 85 kDa, the synthesis of which was sensitive to tetracycline, but not cycloheximide. It is concluded that tetracycline exerts its action through the effect on parasite mitochondria and mitochondrial protein synthesis.

2,4-Dinitrophenol↗

PubMed1989

High-performance liquid chromatographic assay for thymidylate synthase from the human malaria parasite, Plasmodium falciparum.

A rapid and highly sensitive high-performance liquid chromatographic assay for thymidylate synthase activity is described. The assay is based on the separation of the substrate, deoxyuridylate (dUMP), and its product, deoxythymidylate (dTMP), on a LiChrosorb RP-8 reversed-phase column with 44 mM triethylammonium phosphate (pH 7.0) as mobile phase and a flow-rate of 1.0 ml/min. In addition, using a mu Bondapak C18 reversed-phase column with 10 mM potassium phosphate (pH 4.0) and a gradient of 0-28% methanol, dUMP, dTMP and deoxythymidine (dTdR) are well separated within 30 min. The latter system is also applied to assay thymidine kinase activity with dTdR and dTMP as substrate and product, respectively. This method is sensitive enough to measure dTMP at concentrations as low as 25 pmol, and it was used to show that crude extracts of the human malaria parasite Plasmodium falciparum contain thymidylate synthase but not thymidine kinase activity.

Animals↗

PubMed1989

De novo and salvage biosynthesis of pteroylpentaglutamates in the human malaria parasite, Plasmodium falciparum.

Plasmodium falciparum was shown to synthesize pteroylpolyglutamate de novo from guanosine 5'-triphosphate (GTP), p-aminobenzoate (PABA), and L-glutamate (L-Glu). The parasite also had the capacity to synthesize pteroylpolyglutamate from both intact and degradation moieties (p-aminobenzoylglutamate and pterin-aldehyde) of exogenous folate added into the growth medium. The major product was identified as 5-methyl-tetrahydroteroylpentaglutamate following exposure to pteroylpolyglutamate hydrolase and oxidative degradation of the C9-N10 bond in the molecule and identification of products by reversed-phase high performance liquid chromatography. Inhibition of pteroylpentaglutamate synthesis from the radiolabelled metabolic precursors (GTP, PABA, L-Glu) and folate by the antifolate antimalarials, pyrimethamine and sulfadoxine at therapeutic concentrations, may suggest the existence of a unique biosynthetic pathway in the malaria parasite.

Animals↗

PubMed1989

Characterization of cobalamin-dependent methionine synthase purified from the human malarial parasite, Plasmodium falciparum.

Methionine synthase, which catalyzes the reaction, 5-methyltetrahydrofolate (5-CH3-H4PteGlu) + homocysteine----methionine + tetrahydrofolate, was detected and partially purified from the human malarial parasite, Plasmodium falciparum (K1 isolate). Partial purification was achieved using high-performance size-exclusion and anion-exchange chromatography. The apparent relative molecular weight of the enzyme was estimated as 105,000 daltons, and the apparent Km for 5-CH3-H4PteGlu was 24.2 microM. The enzyme was dependent on adenosylcobalamin or methylcobalamin but not on cobalamin, cyanocobalamin, or hydroxocobalamin in either the absence or presence of S-adenosylmethionine. Preincubation with nitrous oxide markedly inhibited the enzyme. Methionine synthase in P. falciparum may play a role in the supply of methionine and in folate salvage using exogenous 5-CH3-H4PteGlu for tetrahydrofolate metabolism.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1989

Susceptibility to hydrogen peroxide of Plasmodium falciparum infecting glucose-6-phosphate dehydrogenase-deficient erythrocytes.

The susceptibility to oxidant-mediated killing of Plasmodium falciparum infecting normal and glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes was assessed by exposure to hydrogen peroxide generated by the glucose-glucose oxidase system. The parasites infecting G6PD-deficient erythrocytes had markedly greater susceptibility to hydrogen peroxide under a variety of conditions than those infecting normal erythrocytes. In both cases, the killing effect was mediated mainly through the host cells since treatment of the erythrocytes with hydrogen peroxide did not change their relatively susceptibility. The parasites were most susceptible during maturation, especially in G6PD-deficient erythrocytes, although a reduction in parasite invasion was also observed. The role of oxidant-mediated killing in the protection of G6PD-deficient hosts from P. falciparum infection is discussed.

Animals↗

PubMed1989

Decreased sensitivity of artesunate and chloroquine of Plasmodium falciparum infecting hemoglobin H and/or hemoglobin constant spring erythrocytes.

Plasmodium falciparum infecting hemoglobin (Hb) H and/or Hb Constant Spring erythrocytes in vitro was relatively more resistant than that infecting normal erythrocytes to artesunate and chloroquine, while the sensitivity to pyrimethamine was unchanged. The 50% inhibitory concentrations (IC50) for artesunate in HbH (alpha-thal 1/alpha-thal 2), HbH (alpha-thal 1/Hb Constant Spring), and homozygous Hb Constant Spring erythrocytes were 4.5 +/- 2.8, 8.5 +/- 3.2, and 2.6 +/- 1.6 nM compared with 0.82 +/- 0.35 nM in normal erythrocytes (P less than 0.002 for all three cases). The IC50 for chloroquine were 97 +/- 46, 162 +/- 67, and 93 +/- 36 nM, respectively, in the variant erythrocytes, compared with 48 +/- 13 nM in normal erythrocytes (P less than 0.002, 0.002, and 0.02, respectively). The differences in sensitivity to artesunate and chloroquine of the parasite infecting HbH erythrocytes are probably related to their oxidative mode of action and relatively high amounts of antioxidant enzymes in the host erythrocytes. This novel example of dependence on the host of the malarial parasite drug sensitivity may have implications for chemotherapy of malaria in patients with genetically variant erythrocytes.

Animals↗