Inhibitory effects of anthracyclines on partially purified 5'-3' DNA helicase of Plasmodium falciparum.

BACKGROUND: Plasmodium falciparum has been becoming resistant to the currently used anti-malarial drugs. Searching for new drug targets is urgently needed for anti-malarial development. DNA helicases separating double-stranded DNA into single-stranded DNA intermediates are essential in nearly all DNA metabolic transactions, thus they may act as a candidate for new drug targets against malarial parasites. METHODS: In this study, a P. falciparum 5' to 3' DNA helicase (PfDH-B) was partially purified from the crude extract of chloroquine- and pyrimethamine-resistant P. falciparum strain K1, by ammonium sulfate precipitation and three chromatographic procedures. DNA helicase activity of partially purified PfDH-B was examined by measuring its ability to unwind 32P-labelled partial duplex DNA. The directionality of PfDH-B was determined, and substrate preference was tested by using various substrates. Inhibitory effects of DNA intercalators such as anthracycline antibiotics on PfDH-B unwinding activity and parasite growth were investigated. RESULTS: The native PfDH-B was partially purified with a specific activity of 4150 units/mg. The PfDH-B could unwind M13-17-mer, M13-31-mer with hanging tail at 3' or 5' end and a linear substrate with 3' end hanging tail but not blunt-ended duplex DNA, and did not need a fork-like substrate. Anthracyclines including aclarubicin, daunorubicin, doxorubicin, and nogalamycin inhibited the unwinding activity of PfDH-B with an IC50 value of 4.0, 7.5, 3.6, and 3.1 µM, respectively. Nogalamycin was the most effective inhibitor on PfDH-B unwinding activity and parasite growth (IC50 = 0.1 ± 0.002 µM). CONCLUSION: Partial purification and characterization of 5'-3' DNA helicase of P. falciparum was successfully performed. The partially purified PfDH-B does not need a fork-like substrate structure found in P. falciparum 3' to 5' DNA helicase (PfDH-A). Interestingly, nogalamycin was the most potent anthracycline inhibitor for PfDH-B helicase activity and parasite growth in culture. Further studies are needed to search for more potent but less cytotoxic inhibitors targeting P. falciparum DNA helicase in the future.

An In Vitro Study on the Effects of Selected Natural Dietary Fiber from Salad Vegetables for Lowering Intestinal Glucose and Lipid Absorption.

BACKGROUND: Salad vegetables are good sources of dietary fiber and are becoming increasingly popular among consumers. Therefore, these plants have the potential to be developed as functional foods. OBJECTIVE: Using an in vitro model, this study investigated the physical properties and intestinal glucose and lipid absorption capacities of dry dietary fiber from vegetables typically consumed in salads (types of lettuce, including red oak, red coral, green oak, butterhead, and cos). METHOD: Fiber was prepared from each type of lettuce using an enzymatic method and then characterized. Physical properties, including solubility and water-binding, swelling, cation-exchange, and oil-binding capacities, and antihyperglycemic and antihypercholesterolemic effects of fiber were investigated. RESULTS: The hydration capacity of total dietary fiber and insoluble fiber from the majority of sources was significantly different from that of cellulose. Adsorption and diffusion of glucose were directly proportional to incubation time, and the diffusion rate was significantly lower in the treatments containing fiber compared to the cellulose control. Fiber from these vegetables also inhibited amylase and alpha-glucosidase activities. Moreover, fiber from all sources exhibited significantly higher sodium cholate and cholesterol-binding capacity compared to cellulose, and also retarded pancreatic cholesterol esterase activity in a concentration-dependent manner. CONCLUSION: This study demonstrates that natural dietary fiber from salad vegetables can reduce glucose and lipid absorption and breakdown rates, thus preventing increases in postprandial blood glucose and cholesterol levels, which can be beneficial to human health.

Study of prebiotic properties of selected banana species in Thailand.

Prebiotics are functional foods with health-promoting properties that are used in many developed countries. Thailand is one of the countries that produces many plants that should have prebiotic properties. In this study, we investigated the potential prebiotic effects of powders obtained from Saba, Pisang Awak Banana and Silver bluggoe in vitro in accordance with their physical, chemical and microbiological properties. These selected plants were found to demonstrate good water-/oil-binding properties. They contained chlorophyll, beta carotene and lycopene and showed good resistance to stomach and small-intestine enzymes. The selected plants were further used to evaluate prebiotic properties by supplementing as a carbon source in culturing broth for growing probiotic bacteria and pathogenetic bacteria. The increase in the number of probiotic bacteria during fermentation of these selected plants correlated with decreased pH. The growth of four strains of probiotic bacteria seemed to be promoted in MRS broth containing these selected plants, but no significant differences in the number of probiotic bacterial groups were detected in response to difference concentrations of all these selected plants. In addition, we noted that a decrease in the number of all four strains of pathogenic bacteria during fermentation of these selected plants correlated with a decreased pH. Moreover, the antimicrobial activity of selected plant prebiotics supported probiotic substance production to inhibit growth of pathogenic bacteria. In conclusion, we have shown that the addition of selected prebiotic plants, indicating that they should be used as a prebiotic food ingredient, represents a potential alternative to available commercial prebiotics.

Characterization of Plasmodium falciparum ATP-dependent DNA helicase RuvB3.

BACKGROUND: Malaria is one of the most serious and widespread parasitic diseases affecting humans. Because of the spread of resistance in both parasites and the mosquito vectors to anti-malarial drugs and insecticides, controlling the spread of malaria is becoming difficult. Thus, identifying new drug targets is urgently needed. Helicases play key roles in a wide range of cellular activities involving DNA and RNA transactions, making them attractive anti-malarial drug targets. METHODS: ATP-dependent DNA helicase gene (PfRuvB3) of Plasmodium falciparum strain K1, a chloroquine and pyrimethamine-resistant strain, was inserted into pQE-TriSystem His-Strep 2 vector, heterologously expressed and affinity purified. Identity of recombinant PfRuvB3 was confirmed by western blotting coupled with tandem mass spectrometry. Helicase and ATPase activities were characterized as well as co-factors required for optimal function. RESULTS: Recombinant PfRuvB3 has molecular size of 59 kDa, showing both DNA helicase and ATPase activities. Its helicase activity is dependent on divalent cations (Cu2+, Mg2+, Ni+2 or Zn+2) and ATP or dATP but is inhibited by high NaCl concentration (>100 mM). PfPuvB3 is unable to act on blunt-ended duplex DNA, but manifests ATPase activity in the presence of either single- or double-stranded DNA. PfRuvB3.is inhibited by doxorubicin, daunorubicin and netropsin, known DNA helicase inhibitors. CONCLUSIONS: Purified recombinant PfRuvB3 contains both DNA helicase and ATPase activities. Differences in properties of RuvB between the malaria parasite obtained from the study and human host provide an avenue leading to the development of novel drugs targeting specifically the malaria form of RuvB family of DNA helicases.

Characterization of recombinant malarial RecQ DNA helicase.

RecQ DNA gene of multi-drug resistant Plasmodium falciparum K1 (PfRecQ1) was cloned, and the recombinant C-terminal-decahistidine-tagged PfRecQ1 was expressed in Escherichia coli. The purified enzyme could efficiently unwind partial duplex DNA substrate in a 3' to 5' direction. The malarial RecQ1 could not unwind substrates with both 5' and 3' overhangs, those with a 5' overhang, or blunt-ended DNA duplexes. Unwinding of DNA helicase activity was driven by the hydrolysis of ATP. The drug inhibitory effects of six compounds indicated that only doxorubicin and daunorubicin could inhibit the unwinding activity.

Anisakids in marine fish from the coast of Chon Buri Province, Thailand.

A total of 1,600 specimens, consisting of 16 different species of marine fish, were dissected and examined for anisakid larvae and adults in visceral organs, abdominal cavity, and muscles. One species of adult-stage nematode was found in two of 16 species of marine fish studied, Johnius carouna and Dendrophysa russelli. No anisakid larvae (third-stage) was found in any of the 16 species of marine fish studied. Morphological study of the adult-stage nematode showed similar morphology to Anisakis simplex. We found that the nematode adult recovered from the marine fish differed from other anisakids in morphology, life cycle and locality of infection in the fish. The anisakid adults recovered were ovoviviparous or larviparous, but not oviparous as is seen in most other anisakids. The intensity and prevalence of nematode infection in Johnius carouna were 2.4 and 31.7%, respectively, and in Dendrophysa russelli 3.9 and 87.5%, respectively.