Abstract: The expression profiles of nine genes encoding chitin deacetylase (CDA)-like proteins were studied during development and in various tissues of the red flour beetle, Tribolium castaneum, by RT-PCR. TcCDA1, TcCDA2 and TcCDA5 were expressed throughout all stages of development, while TcCDA6-9 were expressed predominantly during larval feeding stages. In situ hybridization experiments revealed that both TcCDA1 and TcCDA2 were expressed in epidermal cells. Polyclonal antibody to TcCDA1 detected an immunoreactive protein in larval tracheae. TcCDA6 through TcCDA9, which belong to a distinct subgroup of gut-specific CDAs, were transcribed in the cells lining the midgut, including epithelial cells. TcCDA3 was expressed in the thoracic muscles, whereas TcCDA4 was expressed in early imaginal appendages. To study the function(s) of individual TcCDA genes, double-stranded RNAs (dsRNA) specific for each gene were injected into insects at different developmental stages and the phenotypes were monitored. No visible phenotypic changes were observed after injection of dsRNAs for TcCDA3 to 9, whereas injection of dsRNAs for TcCDA1 or TcCDA2 affected all types of molts, including larval-larval, larval-pupal and pupal-adult. Insects treated with these dsRNAs could not shed the old cuticle and were trapped in their exuviae. Interestingly, unique and very dissimilar adult phenotypes were observed after injection of dsRNAs that specifically down-regulated either of the two alternatively spliced transcripts of TcCDA2, namely TcCDA2a or TcCDA2b. These results reveal functional specialization among T. castaneum CDA genes and splice variants.
Abstract: Ion transport peptide (ITP) and ITP-like (ITPL) are highly conserved neuropeptides in insects and crustaceans. We investigated the alternatively spliced variants of ITP/ITPL in Tribolium castaneum to understand their functions. We identified three alternatively spliced transcripts named itp, itpl-1, and itpl-2. Expression patterns of the splice variants investigated by exon-specific in situ hybridization were somewhat different from those previously reported in other insect species. Most importantly, we found for the first time that itpl-1 transcripts are abundantly expressed in the midgut at the late larval stage, showing an expression pattern similar to that of the crustacean hyperglycemic hormone (CHH) in the crab Carcinus maenas. CHH was shown to function by increasing the body volume through fluid absorption, resulting in breakage of the outer shell at the time of molt. Exon-specific RNA interference (RNAi) was designed to distinguish between itp and itpl-1, but we were unable to design a dsRNA uniquely targeting or uniquely excluding itpl-2; therefore, RNAi targeting was limited to either itp/itpl-2 or itpl-1/itpl-2. For dsRNA injections in the larval stages, either RNAi led to gradually increasing mortality in the larval and pupal stages, with 100% cumulative mortality at the time of eclosion or shortly afterward. Developmental deficiencies in the adult tarsal segments were observed after RNAi suppressing either itp/itpl-2 or itpl-1/itpl-2. After dsRNA injections at the pupal stage, the most striking observation was a significant reduction in egg numbers (8% of control) and reduced survival of the offspring (5%) in RNAi targeting itpl-1/itpl-2, while a milder degree of the same phenotype was observed in that targeting itp/itpl-2.
Abstract: Atrazine is an extensively used triazine herbicide in agricultural and residential areas and has been routinely detected in many surface and ground waters. This study reveals various up- and down-regulated genes associated with hypoxic stress in atrazine-treated fourth-instar Chironomus tentans larvae (midges) by using restriction fragment differential display-PCR. Two down-regulated hemoglobin cDNAs were isolated from the midges. Northern blot analysis indicated CteHb-IIbeta and CteHb-III mRNA expressions decreased by 36 and 21%, respectively, in midges exposed to atrazine at 1mug/L for 96h. Decreased hemoglobin gene expression was associated with elevated oxygen consumption in atrazine-treated midges. Midges exposed to atrazine at 1mug/L increased their oxygen consumption by 47%, whereas midges exposed to atrazine at 1000mug/L for 48h increased their oxygen consumption by 66%. Our study demonstrates for the first time that atrazine, at environmentally relevant concentrations, can elevate respiration, possibly eliciting counteractive measures at the transcriptional level to adapt to oxygen deficiency in an ecologically important aquatic insect. Our results further suggest that the ability to modulate both the quantity and quality of Hb serves as an adaptive response to counteract the initial onset of oxygen deficiency induced by atrazine in midges.
Abstract: Quinazolinone type alkaloids, febrifugine (1) and isofebrifugine (2), isolated from Dichroa febrifuga roots, show powerful antimalarial activity against Plasmodium falciparum. Unfortunately, their emetic effect and other undesirable side effects have precluded their clinical use for malaria. Because of their antimalarial potency, analogues were searched for, with the goal of preserving the strong antimalarial activity, while dramatically reducing side effects. We expected that compounds useful in drug development would exist in metabolites derived from 1 and Df-1 (3), the condensation product of 1 with acetone, by mouse liver S9. Feb-A and -B (4 and 5) were isolated as the major metabolites of 1. In addition to 4 and 5, feb-C and -D (6 and 7) were also purified from the metabolic mixture of 3. Compounds 4 and 5 were compounds oxidized at C-6 and C-2 of the quinazolinone ring of 1, respectively. Compounds 6 and 7, derived from 3, also bear febrifugine type structures in which the 4' '- and 6' '-positions of the piperidine ring of 1 were oxidized. In vitro antimalarial and cytotoxic tests using synthetically obtained racemic 4-6 and enantiomerically pure 7 demonstrated that 4 and 6 had antimalarial activity against P. falciparum, of similar potency to that of 1, with high selectivity. The antimalarial activity of 5 and 7, however, was dramatically decreased in the test. The in vitro antimalarial activity of analogues 22 and 43, which are stereoisomers of 4 and 6, was also evaluated, showing that 22 is active. The results suggest that basicity of both the 1- and the 1' '-nitrogen atoms of 1 is crucial in conferring powerful antimalarial activity. Racemic 4 and 6 exhibited powerful in vivo antimalarial activity against mouse malaria P. berghei, and especially, no serious side effects were observed with 4. Thus, the metabolite 4 appears to be a promising lead compound for the development of new types of antimalarial drugs.
Abstract: On the basis of a comparative analysis for stability in mouse serum between 15-O-acetylbruceolide and bruceolide 15-methyl carbonate, several 3,15-dialkyl carbonates of bruceolide were synthesized and their in vitro antimalarial activity was assessed. Methyl, ethyl, and isopropyl carbonates with pronounced in vitro activity were further evaluated for in vivo antimalarial potency. Both the methyl and ethyl carbonates significantly increased the life span of mice as compared with 3,15-di-O-accetylbruceolide and chloroquine.
Abstract: The in vitro antimalarial activity against Plasmodium falciparum and heme polymerization were evaluated for ten metalloporphyrins: gallium protoporphyrin IX (GaPPIX), sodium salt of gallium protoporphyrin IX, silver protoporphyrin IX, palladium protoporphyrin IX, cobalt protoporphyrin IX, manganese protoporphyrin IX, tin protoporphyrin IX (SnPPIX), chromium protoporphyrin IX, gallium deuteroporphyrin IX (GaDPIX) and gallium hematoporphyrin IX. Metalloporphyrins inhibited parasite growth with 50% inhibitory concentrations (IC(50)) ranging from 15.5 microM to 190 microM. In trophozoite lysate-mediated heme polymerization assays, SnPPIX, GaPPIX and GaDPIX exerted potent inhibitory activity similar to that of artemisinin and chloroquine.
Abstract: Co(II)-catalyzed peroxidation of dienes including (S)-limonene in the presence of molecular oxygen and triethylsilane provided in each case the corresponding 2,3-dioxabicyclo[3.3.1]nonane derivatives via the intramolecular cyclization of the unsaturated peroxy radical intermediates. The product composition was remarkably influenced by the structure of the dienes, the nature of the solvents, and the concentration of the substrates and the catalyst. Some of the yingzhaosu A analogues obtained in this study showed notable antimalarial activities in vitro.
Abstract: Photooxygenation of 2-phenylnorbornene 1 in the presence of 30% aqueous hydrogen peroxide afforded 1,2-bishydroperoxide 3, which could be cycloalkylated on treatment with silver oxide and a 1,omega-diiodoalkane to provide the tricyclic peroxides 12. Trimethylsilylation of 3 followed by TMSOTf-catalyzed cyclocondensation with carbonyl compounds led to the formation of the tricyclic peroxides 14 containing a 1,2,4,5-tetroxepane structure. Photooxygenation of 1 in the presence of either unsaturated hydroperoxides or unsaturated alcohols followed by bis(collidine)iodine hexafluorophosphate promoted cyclization gave the corresponding cyclic peroxides 15-17. Several of these cyclic peroxides showed substantial antimalarial activity particularly in vitro.
Abstract: Among 42 extracts, prepared from 14 medicinal plants used in Vietnamese traditional medicine to treat malaria, 24 were found to have antiplasmodial activity by inhibiting the growth of the chloroquine-resistant Plasmodium falciparum strain FCR-3 with EC(50) values less than 10 microg/ml. Each medicinal plant possessed at least one active extract. The methanol extract of Coscinium fenestratum had the strongest antiplasmodial activity with EC(50) value of 0.5 microg/ml. Activity-guided fractionation led to identification of berberine as the major active constituent.
Abstract: Iodonium ion mediated cyclization of unsaturated hydroperoxides 1 afforded the expected yingzhaosu A analogues 2. In some cases, however, the corresponding cyclic ethers 5 were formed competitively with the cyclic peroxides 2, the ratios of these two products being a marked function of the structure of the starting materials. Some of the cyclic peroxides 2 showed significant antimalarial activities in vitro and in vivo.
Abstract: We studied sequence polymorphism, expression level of pfmdr1 gene and drug sensitivity in mefloquine sensitive-strain as well as mefloquine-resistant clone/24 to understand underlying drug resistance mechanism. Mefloquine-resistant clone/24 exhibited decreased susceptibility to mefloquine, quinine, halofantrine and artemisinin and increased susceptibility to chloroquine. We analyzed sequence of pfmdr1 gene and found mutation in pfmdr1 on clone/24. Moreover, overexpression of mRNA level of pfmdr1 has been observed in mefloquine-resistant clone/24. Our results explain gene mutation and expression level of pfmdr1 may be associated in mefloquine resistance mechanism.
Abstract: Drug resistance in Plasmodium falciparum is a serious problem in most endemic areas. Recent studies have suggested the potential involvement of genes in the MDR gene family in resistance to quinoline-containing compounds in P. falciparum. In our present studies, a molecular analysis of pfmdr 1 in isolate strain of P. falciparum, 523a R, from Japanese mefloquine-resistant patient was done to determine the reported association of pfmdr 1 intragenic alleles and mefloquine resistance, and to examine the antimalarial activities of several antimalarial agents against the P. falciparum strain. The antimalarial activities against the strain was decreased susceptibility to mefloquine, artemisinin and halofantrine, in contrast increased susceptibility to chloroquine. The DNA sequence analysis of pfmdr 1 gene in a strain reveled no association of intragenic alleles with mefloquine resistance. Furthermore, the overexpression of pfmdr1 mRNA have been observed and it is about 7.2 times higher than sensitive strain. Our data shows that overexpression of pfmdr1 gene may be associated in mefloquine-resistance mechanism.
Abstract: CsOH- or Ag(2)O-mediated cycloalkylation of (alkylidene)bisperoxides 3 and 1,n-dihaloalkanes (n = 3-8) provided the corresponding medium-sized 1,2,4,5-tetraoxacycloalkanes 4-8 in moderate yields. Subsequent evaluation of the antimalarial activity of the cyclic peroxides 4-8 in vitro and in vivo revealed that 1,2,6,7-tetraoxaspiro[7.11]nonadecane 4a has considerable potential as a new, inexpensive, and potent antimalarial drug.
