Plasmodium vinckei: selection of a strain exhibiting stable resistance to arteether

A strain of rodent malaria parasite Plasmodium vinckei showing >12-fold resistance to arteether has been selected after exposure to sub-curative doses of drug in 44 sequential passages over a period of 700 days. Experimentally induced resistance was found to be stable after drug free maintenance of parasites for 11 serial passages over a period of 100 days. Cross-sensitivity studies have shown that apart form resistance to related derivatives like artemether and artesunic acid, the derived parasites also show resistance to quinine and mefloquine.     

苗期紫花苜蓿株体对不同地区垂穗披碱草种子萌发生长的化感作用

以发芽率、苗长、根长、苗干重、根干重变化为种子萌发和幼苗生长参数,研究苗期紫花苜蓿(Medicago sativa)植株浸提液对不同地区垂穗披碱草(Elymus nutans)种子萌发生长的化感作用。结果表明:地上部浸提液对LS、DX垂穗披碱草种子发芽率具有明显的促进作用,而对GS、KMX、LKZ、LZ地区垂穗披碱草种子发芽率均表现为抑制作用,其中对GS垂穗披碱草种子发芽率的抑制作用最强,在14.5%和5.5%浓度处理时抑制率分别为57.89%、55.26%;苗长方面,浸提液5.5%浓度对垂穗披碱草苗长的抑制率顺序为:LZNQDXLSKMXQHGSLKZ,14.5%处理时抑制率顺序为:LZKMXLKZNQGSQHDXLS,其中抑制率最高的为LZ垂穗披碱草,在14.5%和5.5%浓度处理时抑制率分别为33.03%、28.97%;根长方面,5.5%处理对垂穗披碱草根长的抑制率顺序为:QHNQLZKMX、GSLKZDX、LS,14.5%处理时抑制率顺序为:GSQH、NQLZLSKMXDXLKZ,其中在高浓度下抑制率最高的为GS垂穗披碱草,抑制率为57.69%;地上部浸提液对LKZ、LZ垂穗披碱草苗干重均具有促进作用,高浓度浸提液对NQ垂穗披碱草苗干重产生促进作用(RI0),而对KMX、DX垂穗披碱草苗干重均表现为抑制作用;根干重方面,浸提液对LS、QH、GS、NQ、LKZ垂穗披碱草根干重均有明显的抑制作用,而对KMX、DX垂穗披碱草根干重产生促进作用,高浓度浸提液对LZ垂穗披碱草的根干重产生促进作用。从根浸提液的作用来看,根浸提液除对LS、DX垂穗披碱草种子发芽率和根干重、GS垂穗披碱草种子发芽率和苗干重及NQ垂穗披碱草根干重具有促进作用外(P 0.05),对其余地区垂穗披碱草的各项指标均有明显的抑制作用(RI0)。所有以上结果表明,紫花苜蓿植株浸提液对垂穗披碱草种子萌发生长的作用具有一定的浓度效应。不同地区垂穗披碱草对紫花苜蓿地上部浸提液的敏感性趋势总体为:GSQHLSKMXNQLKZLZ,最不敏感或有促进作用的是DX垂穗披碱草;对根浸提液的敏感性趋势总体为:QHNQLZKMX,根浸提液对LS、GS、LKZ、DX垂穗披碱草种子萌发生长具有促进作用。紫花苜蓿植株不同部位浸提液对垂穗披碱草种子萌发生长的化感效应顺序为:地上部根。     

Novel squaramides with in vitro liver stage antiplasmodial activity

A structure–activity relationship study was performed with ten 8-aminoquinoline-squaramides compounds active against liver stage malaria parasites, using human hepatoma cells (Huh7) infected by Plasmodium berghei parasites. In addition, their blood-schizontocidal activity was assessed against chloroquine-resistant W2 strain Plasmodium falciparum. Compound 3 was 7.3-fold more potent than the positive control primaquine against liver-stage parasites, illustrating the importance of the squarate moiety to activity.     

The interplay between drug resistance and fitness in malaria parasites

Controlling the spread of antimalarial drug resistance, especially resistance of Plasmodium falciparum to artemisinin‐based combination therapies, is a high priority. Available data indicate that, as with other microorganisms, the spread of drug‐resistant malaria parasites is limited by fitness costs that frequently accompany resistance. Resistance‐mediating polymorphisms in malaria parasites have been identified in putative drug transporters and in target enzymes. The impacts of these polymorphisms on parasite fitness have been characterized in vitro and in animal models. Additional insights have come from analyses of samples from clinical studies, both evaluating parasites under different selective pressures and determining the clinical consequences of infection with different parasites. With some exceptions, resistance‐mediating polymorphisms lead to malaria parasites that, compared with wild type, grow less well in culture and in animals, and are replaced by wild type when drug pressure diminishes in the clinical setting. In some cases, the fitness costs of resistance may be offset by compensatory mutations that increase virulence or changes that enhance malaria transmission. However, not enough is known about effects of resistance mediators on parasite fitness. A better appreciation of the costs of fitness‐mediating mutations will facilitate the development of optimal guidelines for the treatment and prevention of malaria.     

Gametocytocidal activity of pyronaridine and DNA topoisomerase II inhibitors against multidrug-resistant Plasmodium falciparum in vitro

Gametocytocidal activities of pyronaridine and DNA topoisomerase II inhibitors against two isolates of multidrug-resistant Plasmodium falciparum, KT1 and KT3 were determined. After sorbitol treatment, pure gametocyte cultures of Plasmodium falciparum containing mostly young gametocytes (stage II and III) obtained on day 11 were exposed to the drugs for 48 h. The effect of the drugs on gametocyte development was assessed by counting gametocytes on day 15 of culture. Pyronaridine was the most effective gametocytocidal drug against P. falciparum isolates KT1 and KT3 with 50% inhibitory concentration of 6 and 20 nM, respectively. Moreover, the 50% inhibitory concentration of pyronaridine was lower than that of primaquine which is the only drug used to treat malaria patients harboring gametocytes. Prokaryotic (norfloxacin) and eukaryotic (amsacrine and etoposide) DNA topoisomerase II inhibitors were only effective against asexual but not sexual stages of the malaria parasites. Pyronaridine has both schizontocidal and gametocytocidal activities against the human malaria parasite, P. falciparum.     

Plasmodium relictum (lineage SGS1) and Plasmodium ashfordi (lineage GRW2): the effects of the co-infection on experimentally infected passerine birds

The effects of avian malaria parasites of the genus Plasmodium on their hosts are insufficiently understood. This is particularly true for malarial co-infections, which predominant in many bird populations. We investigated effects of primary co-infection of Plasmodium relictum (lineage SGS1) and Plasmodium ashfordi (GRW2) on experimentally infected naive juveniles of siskin Spinus spinus, crossbill Loxia curvirostra and starling Sturnus vulgaris. All siskins and crossbills were susceptible but starlings resistant to both these infections. A general pattern of the co-infections was that heavy parasitemia (over 35% during peaks) of both parasites developed in both susceptible host species. There were no significant effects of the co-infections on mean body mass of the majority of infected birds. Mean haematocrit value decreased approximately 1.5 and 3 times in siskins and crossbills at the peak of parasitemia, respectively. Mortality was recorded among infected crossbills. We conclude that co-infections of P. relictum and P. ashfordi are highly virulent and act synergetically during primary infections in some but not all passerine birds.     

NMR and crystallographic structures of the FK506 binding domain of human malarial parasite Plasmodium vivax FKBP35

The emergence of drug‐resistant malaria parasites is the major threat to effective malaria control, prompting a search for novel compounds with mechanisms of action that are different from the traditionally used drugs. The immunosuppressive drug FK506 shows an antimalarial activity. The mechanism of the drug action involves the molecular interaction with the parasite target proteins PfFKBP35 and PvFKBP35, which are novel FK506 binding protein family (FKBP) members from Plasmodium falciparum and Plasmodium vivax, respectively. Currently, molecular mechanisms of the FKBP family proteins in the parasites still remain elusive. To understand their functions, here we have determined the structures of the FK506 binding domain of Plasmodium vivax (PvFKBD) in unliganded form by NMR spectroscopy and in complex with FK506 by X‐ray crystallography. We found out that PvFKBP35 exhibits a canonical FKBD fold and shares kinetic profiles similar to those of PfFKBP35, the homologous protein in P. falciparum, indicating that the parasite FKBP family members play similar biological roles in their life cycles. Despite the similarity, differences were observed in the ligand binding modes between PvFKBD and HsFKBP12, a human FKBP homolog, which could provide insightful information into designing selective antimalarial drug against the parasites.     

New permeability pathways induced by the malarial parasite in the membrane of its host erythrocyte: Potential routes for targeting of drugs into infected cells

Malarial parasites propagate asexually inside the erythrocytes of their vertebrate host. Six hours after invasion, the permeability of the host cell membrane to anions and small nonelectrolytes starts to increase and reaches its peak as the parasite matures. This increased permeability differs from the native transport systems of the normal erythrocyte in its solute selectivity pattern, its enthalpy of activation and its susceptibility to inhibitors, suggesting the appearance of new transport pathways. A biophysical analysis of the permeability data indicates that the selectivity barrier discriminates between permeants according to their hydrogen bonding capacity and has solubilization properties compared to those ofiso-butanol. The new permeability pathways could result from structural defects caused in the host cell membrane by the insertion of parasite-derived polypeptides. It is suggested that the unique transport properties of the new pathways be used to target drugs into infected cells, to affect the parasite either directly or through the modulation of the intraerythrocytic environment. The feasibility of drug targeting is demonstrated inin vitro cultures of the human malarial parasitePlasmodium falciparum.     

Plasmodium berghei ANKA: Selection of resistance to piperaquine and lumefantrine in a mouse model

We have selected piperaquine (PQ) and lumefantrine (LM) resistant Plasmodium berghei ANKA parasite lines in mice by drug pressure. Effective doses that reduce parasitaemia by 90% (ED₉₀) of PQ and LM against the parent line were 3.52 and 3.93 mg/kg, respectively. After drug pressure (more than 27 passages), the selected parasite lines had PQ and LM resistance indexes (I₉₀) [ED₉₀ of resistant line/ED₉₀ of parent line] of 68.86 and 63.55, respectively. After growing them in the absence of drug for 10 passages and cryo-preserving them at −80 °C for at least 2 months, the resistance phenotypes remained stable. Cross-resistance studies showed that the PQ-resistant line was highly resistant to LM, while the LM-resistant line remained sensitive to PQ. Thus, if the mechanism of resistance is similar in P. berghei and Plasmodium falciparum, the use of LM (as part of Coartem^®) should not select for PQ resistance.     

Multidrug ATP‐binding cassette transporters are essential for hepatic development of Plasmodium sporozoites

Multidrug resistance‐associated proteins (MRPs) belong to the C‐family of ATP‐binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug‐sensitivity profiles as wild type parasites. We show that MRP1‐deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2‐deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development.     

Preventive and Curative Biological Treatments for Control of Botrytis cinerea Stem Canker of Greenhouse Tomatoes

The effects of biological treatments with PlantShield^®, Prestop^®, Quadra 136, RootShield^®, and S33 (Rhodosporidium diobovatum) and chemical treatment with Decree^® applied as a preventive or curative sprays on stem canker caused by Botrytis cinerea on tomato plants grown in sawdust were studied under near-commercial greenhouse conditions. Prestop^® and Decree^®, applied as preventive or curative sprays, PlantShield^® applied as curative spray, and S33 and Q-136 applied as preventive or preventive plus one spray to wounded surface provided season-long protection from B. cinerea stem canker. These treatments also increased fruit yield and decreased the number of dead plants compared with the inoculated control.     

Human p38 mitogen-activated protein kinase inhibitor drugs inhibit Plasmodium falciparum replication

We recently demonstrated that human p38 mitogen-activated protein kinase (MAPK) inhibitors reduced in vitro and in vivo replication of the protozoan parasites Toxoplasma gondii and Encephalitozoon cuniculi. In this study, we assessed the efficacy of five p38 MAPK inhibitors to block the replication of Plasmodium falciparum in human erythrocytes cultured ex vivo and demonstrate that the pyridinylimidazole RWJ67657 and the pyrrolobenzimidazole RWJ68198 reduced P. falciparum replication, yielded trophozoites that were greatly diminished in size at 24 h, and that these two agents interfered with stage differentiation. Interestingly, the chloroquine-resistant strain W2 was significantly more sensitive to these drugs than was the chloroquine-sensitive strain HB3. These results suggest that pyridinylimidazoles and pyrrolobenzimidazoles designed to inhibit human p38 MAPK activation can be developed to treat malaria.     

Fas has a role in cerebral malaria, but not in proliferation or exclusion of the murine parasite in mice

We examined the susceptibility of murine Fas-deficient mutants to malaria infection in order to investigate the role of Fas in an experimental murine model of cerebral malaria (CM). We infected mice of B6 and CBA wild-type and mutant backgrounds with Plasmodium berghei ANKA. The incidence of CM in the mutant mice (B6-lpr, CBA-lpr^cg) was decreased by about 50% compared with wild-type control strains at 2 weeks after infection. We did not observe significant differences of parasitemia during a murine malaria infection with nonlethal Plasmodium yoelii 17XNL between wild-type and lymphoproliferative (lpr) mutant mice of C3H and MRL genetic backgrounds, although B6-lpr mice exhibited significantly higher parasitemia than did B6 mice 12 to 18 days after infection. These results suggest Fas has a possible role in CM but may not play a major role in the proliferation or exclusion of a murine malaria parasite in a nonlethal infection.     

Plasmodium vivax under the microscope: the Aotus model

The Aotus model for vivax malaria is extremely useful both as a source of living parasites in non-endemic areas, and as a model for vaccine and drug development research. Several species of New World primates can be infected with numerous different strains of Plasmodium vivax. This article reviews some aspects of the Aotus model, discusses the frequently observed hematological changes that can confound interpretation of hemogram data during the course of vivax infection, and provides a partial atlas of parasite forms and Aotus nancymai blood cells.     

一株污水源多重耐药大肠杆菌的耐药性检测及全基因组测序分析

【背景】水体环境分布广、流动性强,是耐药菌和耐药基因传播的主要媒介。【目的】了解北方污水厂大肠杆菌携带的耐药基因及可移动遗传元件情况。【方法】从北方污水厂筛选出一株多重耐药大肠杆菌,通过药敏试验进行耐药性检验,采用96孔板法测定菌株的最小抑菌浓度,利用酶标仪探究亚抑菌浓度抗生素对菌株生长的影响,并对菌株进行全基因组测序,对其携带的耐药基因及可移动遗传元件进行预测。【结果】大肠杆菌WEC对四环素、环丙沙星、诺氟沙星和红霉素具有耐药性,亚抑菌浓度的四环素、环丙沙星和诺氟沙星能够延缓或抑制菌株的生长。WEC菌株的基因组中包含一条大小为4 782 114 bp的环状染色体和2个大小分别为60 306 bp (pWEC-1)和92 065 bp (pWEC-2)的环状质粒。菌株共携带129个耐药基因,其中128个位于染色体上,在染色体上预测到原噬菌体、基因岛及插入序列的存在,部分可移动遗传元件携带有耐药基因。质粒pWEC-1中无耐药基因,pWEC-2含有1个耐药基因,在质粒基因组中预测到原噬菌体和插入序列。【结论】污水源大肠杆菌WEC是一株多重耐药菌株,其基因组中携带耐药基因和多种可移动遗传元件...     

Monooxygenase Activity in Methidathion Resistant and Susceptible Populations of Amblyseius womersleyi (Acari: Phytoseiidae)

The purpose of this study was to evaluate the cytochrome P450-dependent monooxygenase activities in methidathion resistant and susceptible strains of Amblyseius womersleyi Schicha. Artificial laboratory selections for resistance and susceptibility to methidathion were performed in an organophosphate resistant strain of A. womersleyi (Kanaya strain). Selections for susceptibility were also performed in a susceptible strain of this predaceous mite (Ishigaki Strain). After the selection process, the LC₅₀ of methidathion for the selected strains of A. womersleyi were 816 mg/l (Kanaya R), 4.61 mg/l (Kanaya S) and 1.59 mg/l (Ishigaki S). The monooxygenase activities were determined biochemically by the O-deethylation of 7-ethoxycoumarin (7-EC). The monooxygenase activity in adult females of Kanaya R strain (51.1 pmol/30 min/mg protein) was 3.60- and 5.42-fold higher than the activity observed for Kanaya S and Ishigaki S strains, respectively. Significant correlation between monooxygenase activity and LC₅₀ (mg/l) of methidathion was observed analyzing 16 populations of A. womersleyi with different susceptibilities to methidathion. Monooxygenase activity was also evaluated in different life stages (egg, larva, protonymph, deutonymph and adult) of A. womersleyi. The lowest activity was observed for the larval stage, which presented the highest susceptibility to methidathion. Protonymph, deutonymph and adult presented the highest monooxygenase activities. These stages were the most tolerant to methidathion. Monooxygenase activities of the Kanaya R strain were higher than of the Kanaya S strain in all developmental stages. The present study can be helpful for the implementation of a program involving release of insecticide-resistant populations of A. womersleyi in the field. The monooxygenase activity determination is easier and quicker than the estimation of LC₅₀, requiring fewer mites.     

Double-drug development against antioxidant enzymes from Plasmodium falciparum

Abstract

New drugs against malaria are urgently and continuously needed. Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems. A most important antioxidative system consists of (di)thiols which are recycled by disulfide reductases (DR), namely both glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and man, and the thioredoxin reductase (TrxR) of P. falciparum. The aim of our interdisciplinary research is to substantiate DR inhibitors as antimalarial agents. Such compounds are active per se but, in addition, they can reverse thiol-based resistance against other drugs in parasites. Reversal of drug resistance by DR inhibitors is currently investigated for the commonly used antimalarial drug chloroquine (CQ). Our recent strategy is based on the synthesis of inhibitors of the glutathione reductases from parasite and host erythrocyte. With the expectation of a synergistic or additive effect, double-headed prodrugs were designed to be directed against two different and essential functions of the malarial parasite P. falciparum, namely glutathione regeneration and heme detoxification. The prodrugs were prepared by linking bioreversibly a GR inhibitor to a 4-aminoquinoline moiety which is known to concentrate in the acidic food vacuole of parasites. Drug-enzyme interaction was correlated with antiparasitic action in vitro on strains resistant towards CQ and in vivo in Plasmodium berghei-infected mice as well as absence of cytotoxicity towards human cells. Because TrxR of P. falciparum was recently shown to be responsible for the residual glutathione disulfide-reducing capacity observed after GR inhibition in P. falciparum, future development of antimalarial drug-candidates that act by perturbing the redox equilibrium of parasites is based on the design of new double-drugs based on TrxR inhibitors as potential antimalarial drug candidates.     

Novel 2H-isoquinolin-3-ones as antiplasmodial falcipain-2 inhibitors

A series of 1-aryl-6,7-disubstituted-2H-isoquinolin-3-ones (2–10) was synthesized and evaluated for their inhibition against Plasmodium falciparum cysteine protease falcipain-2, as well as against cultured P. falciparum strain FCBR parasites. All compounds displayed inhibitory activity against recombinant falcipain-2 and against in vitro cultured intraerythrocytic P. falciparum, with the exception of 9. The new compounds exhibited no selectivity against human cysteine proteases such as cathepsins B and L. The inhibitory activity of the synthesized compounds was also evaluated against another protozoal cysteine protease, namely rhodesain of Trypanosoma brucei rhodesiense.     

Systematic analysis of HSP gene expression and effects on cell growth and survival at high hydrostatic pressure in Saccharomyces cerevisiae

We systematically investigated the role of HSP genes in the growth and survival of Saccharomyces cerevisiae under high hydrostatic pressure together with analysis of pressure-regulated gene expression. Cells of strain BY4742 were capable of growth at moderate pressure of 25 MPa. When pressure of 25 MPa was applied to the cells, the expression of HSP78, HSP104, and HSP10 was upregulated by about 3- to 4-fold, and that of HSP32, HSP42, and HSP82 was upregulated by about 2- to 2.6-fold. However, the loss of one of the six genes did not markedly affect growth at 25 MPa, while the loss of HSP31 impaired high-pressure growth. These results suggest that Hsp31 plays a role in high-pressure growth but that the six upregulated genes do not. Extremely high pressure of 125 MPa decreased the viability of the wild-type cells to 1% of the control level. Notably, the loss of HSP genes other than HSP31 enhanced the survival rate by about fivefold at 125 MPa, suggesting that the cellular defensive system against high pressure could be strengthened upon the loss of the HSP genes. In this paper, we describe the requirement for and significance of a subset of HSP genes in yeast cell growth at moderate pressure and survival at extremely high pressure.