Key problems in science and technology in Thailand.

Science and technology can make substantial contributions to socio-economic development in Thailand. In order to promote such contributions, problem areas and specific problems of high priority need to be defined, and possible solutions outlined. Important criteria for selecting priority areas and key problems include relevance to development, the availability of human resources and the possibility of their development, the cutting-edge advantage to be gained by development, the possibility of international collaboration, and the possibility of integration with culture and environment. These criteria suggest in Thailand a focus on the development of bioscience and biotechnology, materials science, electronics, and information science.

Avian influenza A/H5N1 neuraminidase expressed in yeast with a functional head domain.

The study reports heterologous expression in Pichia pastoris of active neuraminidase derived from avian influenza virus A/Viet Nam/DT-036/2005(H5N1). A gene encoding the neuraminidase N1 head domain (residues 63-449) was fused directly in-frame with the Saccharomyces cerevisiae alpha-factor secretion signal in pPICZ(A vector. Recombinant N1 neuraminidase was expressed in P. pastoris as a 72kDa secreted, soluble protein. Glycopeptidase F treatment generated a 45kDa product, indicating that the secreted recombinant N1 neuraminidase is an N-linked glycoprotein. Kinetic studies and inhibition tests with oseltamivir carboxylate demonstrated that the recombinant N1 neuraminidase has similar K(m) and K(i) values to those of the viral N1 neuraminidase. This yeast-based heterologous expression system provided functionally active recombinant N1 neuraminidase that should be useful in anti-influenza drug screening, and also as a potential protein-based vaccine.

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.

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.

Rate constants of individual steps in papain-catalysed reactions.

Rate constants for acylation of papain (EC 3.4.22.2) by specific substrates and its subsequent deacylation are derived from kinetic analysis of the reactions in the presence of aminoacetonitrile and methanol. Methyl and ethyl hippurate and methyl N-benzyloxycarbonylglycinate have marginally higher values of rate constants for acylation than for deacylation, while the reverse is true for ethyl N-benzoyl-L-arginate. Both acylation and deacylation are rate-determining for these substrates, while only deacylation irate-determining for methyl-N-acetyl-L-phenylalanylglycinate. Deacylation is the only rate-determining step for p-nitrophenyl esters of hippuric acid, N-benzyloxycarbonylglycine and N-acetyl-L-phenylalanylglycine. These results are discussed in relation to those from inactivation of the enzyme by alkylating agent in the presence of substrate.

The relationship of phosphorylation of membrane proteins with the osmotic fragility and filterability of Plasmodium berghei-infected mouse erythrocytes.

Membrane from Plasmodium berghei-infected mouse erythrocytes showed a pattern of protein phosphorylation which was substantially altered from the normal pattern, with an increase in the phosphorylation of the protein with an apparent molecular weight of 43,000 (M 43), which increased from undetectable in uninfected cells to a maximum in the mature trophozoite stage. Phosphorylation levels of this and other minor bands were strongly correlated with osmotic fragility and filterability. The level of M 43 phosphorylation in membranes from cells which remained intact in a hypotonic medium was 3.82 +/- 0.59-times that of lysed cells, compared with the value of 0.76 +/- 0.07 calculated from distribution alone. Results found when intact erythrocytes were phosphorylated by incubation with [32P]Pi prior to partial lysis were similar to those found when membranes from the lysed and unlysed fractions were subsequently phosphorylated with [gamma-32P]ATP. Infected erythrocytes which could pass repeatedly through 3-micron polycarbonate filters had a much higher phosphorylation level for the M 43 region than whole infected cells with similar parasitemia and stage distribution. The phosphorylation change could play a role in the control of osmotic and mechanical properties of the infected erythrocytes during maturation.

Some chemical characteristics of dimethylsuberimidate and its effect on sarcoplasmic reticulum vesicles.

Sarcoplasmic reticulum vesicles treated with dimethylsuberimidate lose the capacity for ATP-promoted Ca2+ accumulation and show other properties indicative of leaky vesicles. As an aid to assessing whether this effect was caused by cross-linking or by hydrolysis products, characteristics of dimethylsuberimidate hydrolysis under incubation conditions used were measured. At pH 7.0, 25 DEGREES C, dimethylsuberimidate is hydrolyzed with an apparent first order rate constant of 0.016 min-1, to give dimethylsuberate as the principal product. The effect on ATP-promoted Ca2+ accululation was shown to be caused by partially and fully hydrolyzed products of the diimido ester, and not to cross-linking of membrane components.

A simple dual selection for functionally active mutants of Plasmodium falciparum dihydrofolate reductase with improved solubility.

Sufficient solubility of the active protein in aqueous solution is a prerequisite for crystallization and other structural studies of proteins. In this study, we have developed a simple and effective in vivo screening system to select for functionally active proteins with increased solubility by using Plasmodium falciparum dihydrofolate reductase (pfDHFR), a well-known malarial drug target, as a model. Prior to the dual selection process, pfDHFR was fused to green fluorescent protein (GFP), which served as a reporter for solubility. The fusion gene was used as a template for construction of mutated DNA libraries of pfDHFR. Two amino acids with large hydrophobic side chains (Y35 and F37) located on the surface of pfDHFR were selected for site-specific mutagenesis. Additionally, the entire pfDHFR gene was randomly mutated using error-prone PCR. During the first step of the dual selection, mutants with functionally active pfDHFR were selected from two libraries by using bacterial complementation assay. Fluorescence signals of active mutants were subsequently measured and five mutants with increased GFP signal, namely Y35Q + F37R, Y35L + F37T, Y35G + F37L and Y35L + F37R from the site-specific mutant library and K27E from the random mutant library, were recovered. The mutants were expressed, purified and characterized as monofunctional pfDHFR following excision of GFP. Our studies indicated that all mutant pfDHFRs exhibited kinetic properties similar to that of the wild-type protein. For comparison of protein solubility, the maximum concentrations of mutant enzymes prior to aggregation were determined. All mutants selected in this study exhibited 3- to 6-fold increases in protein solubility compared with the wild-type protein, which readily aggregated at 2 mg/ml. The dual selection system we have developed should be useful for engineering functionally active protein mutants with sufficient solubility for functional/structural studies and other applications.

An overview of chemotherapeutic targets for antimalarial drug discovery.

The need for new antimalarials comes from the widespread resistance to those in current use. New antimalarial targets are required to allow the discovery of chemically diverse, effective drugs. The search for such new targets and new drug chemotypes will likely be helped by the advent of functional genomics and structure-based drug design. After validation of the putative targets as those capable of providing effective and safe drugs, targets can be used as the basis for screening compounds in order to identify new leads, which, in turn, will qualify for lead optimization work. The combined use of combinatorial chemistry--to generate large numbers of structurally diverse compounds--and of high throughput screening systems--to speed up the testing of compounds--hopefully will help to optimize the process. Potential chemotherapeutic targets in the malaria parasite can be broadly classified into three categories: those involved in processes occurring in the digestive vacuole, enzymes involved in macromolecular and metabolite synthesis, and those responsible for membrane processes and signalling. The processes occurring in the digestive vacuole include haemoglobin digestion, redox processes and free radical formation, and reactions accompanying haem release followed by its polymerization into haemozoin. Many enzymes in macromolecular and metabolite synthesis are promising potential targets, some of which have been established in other microorganisms, although not yet validated for Plasmodium, with very few exceptions (such as dihydrofolate reductase). Proteins responsible for membrane processes, including trafficking and drug transport and signalling, are potentially important also to identify compounds to be used in combination with antimalarial drugs to combat resistance.

Possible modes of action of the artemisinin-type compounds.

Artemisinin-type compounds are used for the treatment of uncomplicated and severe forms of malaria. They reduce parasitaemia more rapidly than any other antimalarial compound known, and are effective against multidrug-resistant parasites. However, uncertainties remain as to how they act on the parasite and cause toxicity. In this review, we summarize current ideas.

Enhanced Ca2+ uptake by mouse erythrocytes in malarial (Plasmodium berghei) infection.

Erythrocytes from Plasmodium berghei-infected mice on incubation either in plasma or artificial isotonic media showed an increase in uptake of 45Ca2+ compared with erythrocytes from uninfected mice. Infected cells (55% parasitaemia) incubated in plasma from normal or infected mice gave uptake rates of 9.8 and 8.1 nmol h-1 per 10(10) cells, assuming equilibrium between added 45Ca2+ and plasma Ca2+. Uptake rates of erythrocytes from infected mice were increased in the presence of glucose, with a rate of 15.0 nmol h-1 per 10(10) cells (52-58% parasitaemia) at 5 mM glucose, compared with 1.5 nmol h-1 per 10(10) cells in the absence of glucose. The enhancement of 45Ca2+ uptake was more pronounced with increasing parasitaemia, and in the fraction relatively enriched with erythrocytes carrying mature parasites. It is likely, therefore, that the enhancement is due to changes in membrane permeability accompanying parasite development. Enhanced haemolysis accompanied 45Ca2+ uptake of erythrocytes carrying mature parasites, but not of those carrying young parasites or uninfected erythrocytes. The possible role of an altered Ca2+ status in erythrocyte pathophysiology during malarial infection is discussed.

Kinetic and molecular properties of dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium chabaudi.

Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) was partially purified from a cloned strain of pyrimethamine-sensitive Plasmodium chabaudi and a drug-resistant clone derived from it. A molecular weight of approximately 120000 was estimated by gel filtration for enzyme from both pyrimethamine-sensitive and resistant parasites. The specific activities of the crude enzyme at pH 7.4 were 2.7 +/- 0.8 and 1.4 +/- 0.6 nmol min-1 mg-1 protein for sensitive and resistant strains, respectively. Methotrexate titration (pH 7.4, 37 degrees C) indicated that the apparent turnover number of the enzyme from the sensitive parasites was 1229 +/- 322 mol min-1 mol-1 compared with 1238 +/- 179 mol min-1 mol-1 for the enzyme from the resistant parasites. There was therefore no significant difference in the amounts of the enzyme from both sources. The Km value for dihydrofolate (9.3 microM) of the enzyme from the drug-sensitive parasites at pH 7.4 was lower than that from the resistant parasites by a factor of approximately 4. The Km values for NADPH of the enzyme from both sources were similar. Inhibition by pyrimethamine of the enzyme from the sensitive parasites was competitive with dihydrofolate, with Ki of 0.26 nM. By contrast, noncompetitive inhibition was observed for the enzyme from the resistant parasites, with Kis of 50 nM and Kii of 33 nM. The enzyme from drug-sensitive and drug-resistant parasites had different activity profiles with respect to pH and temperature. Moreover, the former was more sensitive to heat denaturation than the latter. From these results, it was concluded that the major basis for drug resistance is not an increase in enzyme content, but a large decrease in drug binding with the structurally different enzyme.

Guanosine triphosphate cyclohydrolase in Plasmodium falciparum and other Plasmodium species.

GTP cyclohydrolase (EC 3.5.4.16), the first enzyme in the pteridine pathway leading to the de novo formation of folic acid, has been identified and isolated from the human malaria parasite, Plasmodium falciparum. The enzyme was purified 200-fold by high performance size-exclusion chromatography on a TSK-G-3000 SW protein column. The molecular weight was estimated at 300 000. Optimal enzyme activity was observed at pH 8.0 and 42 degrees C. The Km for GTP was 54.6 microM. Products of the enzyme reaction were identified as the carbon-8 of GTP and D-erythro-dihydroneopterin triphosphate. ATP was a competitive inhibitor (Ki = 600 microM) of the enzyme. Activity of the enzyme was Mg2+-independent, whereas Mn2+, Cu2+ and Hg2+ (5 mM) were inhibitory. GTP cyclohydrolase activity was also identified in a murine parasite, Plasmodium berghei, and a simian parasite, Plasmodium knowlesi. Activity of the enzyme in P. knowlesi, an intrinsically synchronous quotidian parasite, was found to be dependent on the stage of parasite development.

Depression of Plasmodium falciparum dihydroorotate dehydrogenase activity in in vitro culture by tetracycline.

The activity of Plasmodium falciparum dihydroorotate dehydrogenase, a particulate, electron transport-linked enzyme involved in de novo pyrimidine synthesis, was depressed when the parasite was cultured in the presence of a therapeutic concentration of tetracycline over a 96 h period. There was no direct inhibitory effect of the antibiotic on the enzyme activity. The activity of glutamate dehydrogenase, which is cytoplasmic in the parasite, was unaffected by tetracycline over the same period. Dihydroorotate dehydrogenase activity was substantially recovered when electron acceptors were added. It is suggested that the effect of tetracycline is manifested at the level of the dehydrogenase and/or the electron transport chain linked to this enzyme.

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.

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.

Chemical synthesis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene.

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-known target for pyrimethamine and cycloguanil. The low amounts of enzyme obtainable from parasites or the currently available heterologous expression systems have thus far hindered studies of this enzyme. The 1912-base pair P. falciparum DHFR-TS gene was designed based on E. coli codon preference with unique restriction sites evenly placed throughout the coding sequence. The gene was designed and synthesized as three separated domains: the DHFR domain, the junctional sequence, and the TS domain. Each of these domains contained numerous unique restriction sites to facilitate mutagenesis. The three domains were assembled into a complete DHFR-TS gene which contained 30 unique restriction sites in the coding sequence. The bifunctional DHFR-TS was expressed from the synthetic gene as soluble enzyme in E. coli about 10-fold more efficiently than from the wild-type sequence. The DHFR-TS from the synthetic gene had kinetic properties similar to those of the wild-type enzyme and represents a convenient source of protein for further study. The unique restriction sites in the coding sequence permits easy mutagenesis of the gene which should facilitate further understanding of the molecular basis of antifolate resistance in malaria.

Correlation of antimalarial activity of artemisinin derivatives with binding affinity with ferroprotoporphyrin IX.

The antimalarial activity of a number of artemisinin derivatives, both newly synthesized and currently used as drugs, against Plasmodium falciparum in culture shows a correlation with their affinity of binding with ferroprotoporphyrin IX, as measured from the spectral change of the latter. The new C-16-functionalized artemisinin derivatives were obtained through a novel one-pot synthesis of artemisitene (2) from naturally abundant artemisinin (1), followed by Michael addition with nucleophiles. The correlation points to the biological significance of the interaction of these derivatives with ferroprotoporphyrin IX and may provide a basis for primary screening of peroxidic antimalarials of similar structures.

Mechanism-based development of new antimalarials: synthesis of derivatives of artemisinin attached to iron chelators.

Various derivatives of artemisinin covalently linked to iron chelators were synthesized, and their antimalarial activities were evaluated. Although results show no indication that the presence of an iron chelator in the vicinity of artemisinin potentiates its action, the linked compounds prepared still retain comparable activities to that of artemisinin.

C-16 artemisinin derivatives and their antimalarial and cytotoxic activities: syntheses of artemisinin monomers, dimers, trimers, and tetramers by nucleophilic additions to artemisitene.

Nucleophilic additions of lithium keto and ester enolates and mono- and bifunctional Grignard reagents to artemisitene provided C-16-derived artemisinin monomers, dimers, trimers, and tetramers whose antimalarial and cytotoxic activities have been evaluated.