Effect of artemisinin (qinghaosu) and chloroquine on drug-sensitive and drug-resistant strains of Plasmodium falciparum malaria: use of [2,8-3H]adenosine as an alternative to [G-3H]hypoxanthine in the assessment of in vitro antimalarial activity

Using [G-3H]hypoxanthine uptake as a radioactive indicator for the growth of malarial parasites, we measured the antimalarial activity of artemisinin (Qinghaosu, QHS) against FCMSU1/Sudan strain (chloroquine-sensitive strain) and FCB K+ strain (chloroquine-resistant strain) of Plasmodium falciparum in continuous culture in vitro. The 50% inhibitory concentrations (IC50) for QHS against FCMSU1/Sudan strain and FCB K+ strain were 2.8 X 10(-8) and 3.0 X 10(-8) M, respectively. On the contrary, the response of the two strains to chloroquine was quite different. The IC50 for chloroquine against FCMSU1/Sudan strain was 5.6 ng/ml, whereas that for the FCB K+ strain was 65.6 ng/ml. Therefore, QHS did not appear to exhibit any cross-resistance with chloroquine. If [2,8-3H]adenosine was used as a radioactive precursor instead of [G-3H]hypoxanthine for the determination of antimalarial activity, virtually identical results were obtained. Therefore, [2,8-3H]adenosine can be used as an alternative to [G-3H]hypoxanthine for the assessment of antimalarial action.     

Biotransformation of 2-(4-methoxybenzyl)-l-cyclohexanone by cell cultures ofSolatium aviculare

During the search for compounds with insect juvenile hormone activity, the biotransformation of 2-(4-methoxybenzyl)-l-cyclohexanone, of 2-(4-methoxybenzyl)-l-cyclohexanone ethylene acetal and of both isomers of 2-(4-methoxybenzyl)-l-cyclohexanol by plant cells was examined. The compounds were metabolized by cell suspension culture of Solatium aviculare Forst. The reaction conditions were optimized and the metabolic products isolated and identified. A scheme of biotransformation pathway has been proposed.     

Structures of the tetrahydrogenated menaquinones fromActinomadura angiospora,Faenia rectivirgula,andSaccharothrix australiensis

The structures of the tetrahydrogenated menaquinones fromActinomadura angiospora, Faenia rectivirgula, andSaccharothrix australiensis were determined by mass spectrometry and proton nuclear magnetic resonance spectrometry. The positions of saturation of the tetrahydrogenated menaquinones fromFaenia rectivirgula andSaccharothrix australiensis were units II plus III (counting from the ring system), whereas that ofActinomadura angiospora had units III and VIII hydrogenated. The tetrahydrogenated menaquinones fromFaenia rectivirgula andSaccharothrix australiensis are similar to those characterized from other Gram-positive taxa to date, whereas that fromActinomadura angiospora represents a hitherto unknown isomer.     

Differential expression of the human glucocerebrosidase-coding gene

Gaucher disease is an inborn error of sphingolipid metabolism. It is due to decreased enzymatic activity of glucocerebrosidase (GCase) which causes accumulation of glucocerebrosides, mainly in cells of the reticulo-endothelial system. The disorder is clinically heterogenous and can include central nervous system signs. However, the manifestations of the disease in most cases are restricted to a limited number of cell types and organs. This could be explained by highly differential expression of the human gcs gene. To test this notion, the level of GCase-specific mRNA was determined in different human cell lines by hybridizing Northern blots to a human GCase-specific cDNA probe or by using the RNase protection method. It was found that epithelial cells exhibit high levels of GCase mRNA while skin fibroblasts and promyelocytes show intermediate steady-state levels of this RNA. Macrophages have low steady-state levels of GCase mRNA and in B-cells it is hardly detectable. Moreover, when B-cells or skin fibroblasts were transfected with a vector harbouring the bacterial cat gene coupled to the human gcs gene promoter, the levels of CAT expressed in each cell type were directly correlated to the amount of endogenous GCase RNA. Comparison of the GCase mRNA levels in Gaucher-versus non-Gaucher-derived cells revealed that in Gaucher cells this RNA is always more abundant than in the corresponding non-Gaucher counterparts, suggesting the involvement of a feed-back mechanism sensitive to the levels of actual enzymatic activity.     

Poliovirus proteinase 3C: large-scale expression, purification, and specific cleavage activity on natural and synthetic substrates in vitro.

Proteinase 3C of poliovirus type 2 (Sabin) was expressed at 4% total protein in Escherichia coli. The protein was soluble and could be purified by a simple scheme. It was weakly active on the capsid precursor P1 (expressed in vitro), which contains two cleavage sites. The products of processing P1 were 1ABC and 1D (VP1). The activity was insensitive to Triton X-100. Crude extracts of cells infected with poliovirus type 1 (Mahoney) gave strong processing and yielded 1AB (VP0), 1C (VP3), and 1D in the same assay system but were sensitive to detergent. 3C from cell extracts that was separated from its precursors resembled the recombinant proteinase in its activity. Recombinant 3C cleaved the peptide dansyl-Glu-Glu-Glu-Ala-Met-Glu-Gln-Gly-Ile-Thr-Asn-Lys-NH2 at the Gln-Gly bond. We conclude that 3C is merely the core of the Gln-Gly-cleaving activity which processes P1 in vivo and that there is probably a hydrophobic contact between a larger 3C precursor and its P1 substrate which allows the second processing reaction: 1ABC, 1D----1AB, 1C, 1D.     

Characterization of poliovirus 2A proteinase by mutational analysis: residues required for autocatalytic activity are essential for induction of cleavage of eukaryotic initiation factor 4F polypeptide p220.

The poliovirus proteinase 2A is autocatalytically released from the poliovirus polyprotein by cotranslational cleavage at its own amino terminus, resulting in separation of structural and nonstructural protein precursors. Cleavage is a prerequisite for further processing of the structural protein precursor and consequently for poliovirus encapsidation. A second function of 2Apro is in the rapid shutoff of host cell protein synthesis that occurs upon infection with poliovirus. This is associated with proteolytic cleavage of the p220 component of eukaryotic initiation factor eIF-4F, which is induced but not directly catalyzed by 2Apro. We introduced single-amino-acid substitutions in the 2Apro-coding region of larger poliovirus precursors that were subsequently translated in vitro and thus demonstrated that His-20, Asp-38, and Cys-109 (which constitute the putative catalytic triad) are essential for, and that His-117 is an important determinant of, the autocatalytic activity of 2Apro. This is consistent with the proposal that 2Apro is structurally related to a subclass of trypsinlike serine proteinases. Moreover, 2Apro containing a Cys109Ser substitution retained a small but significant autocatalytic activity. Cleavage of p220 was not induced by those mutants that had reduced proteolytic activity, indicating that the cellular factor that cleaves p220 is probably activated by 2Apro-catalyzed proteolytic cleavage.     

Viral proteases as targets for chemotherapeutic intervention

Many viruses encode proteinases that are essential for infectivity, and are consequently attractive chemotherapeutic targets. The biochemistry and structure of the human immunodeficiency virus proteinase have been characterized extensively, and potent peptide-mimetic inhibitors have been developed. Techniques and strategies used to improve the efficiency of these compounds are likely to be applicable to other viral proteinases.     

A bioluminescent assay for the determination of phosphoenolpyruvate carboxykinase activity in nanogram-sized tissue samples

A highly specific and sensitive assay for the determination of phosphoenolpyruvate carboxykinase (PEPCK) in nanogram-sized tissue samples is described. This test system is based on the stoichiometric transformation of phosphoenolpyruvate into ATP. In a subsequent step ATP is quantified by bioluminescent techniques. The applicability of this assay system is shown by measurements in liver samples with normal and high PEPCK activity levels.     

Production of guanidine-resistant and -dependent poliovirus mutants from cloned cDNA: mutations in polypeptide 2C are directly responsible for altered guanidine sensitivity.

cDNA fragments representing the region in polypeptide 2C containing mutations in a guanidine-resistant or -dependent mutant were cloned into the wild-type background of an infectious clone. Transfection of COS-1 cells with these plasmids yielded viruses that were either completely resistant to 2.0 mM guanidine hydrochloride or dependent on this concentration of drug for growth.