The antimicrobial effects of selenium nanoparticle-enriched probiotics and their fermented broth against Candida albicans

Background

Lactic acid bacteria are considered important probiotics for prevention of some infections. The aim of this work was to investigate the effect of selenium dioxide on the antifungal activity of Lactobacillus plantarum and L. johnsonii against Candida albicans.

Methods

Lactobacillus plantarum and L. johnsonii cells, grown in the presence and absence of selenium dioxide, and their cell-free spent culture media were tested for antifungal activity against C. albicans ATCC 14053 by a hole-plate diffusion method and a time-kill assay.

Results

Both L. plantarum and L. johnsonii reduced selenium dioxide to cell-associated elemental selenium nanoparticles. The cell-free spent culture media, from both Lactobacillus species that had been grown with selenium dioxide for 48 h, showed enhanced antifungal activity against C. albicans. Enhanced antifungal activity of cell biomass against C. albicans was also observed in cultures grown with selenium dioxide.

Conclusions

Selenium dioxide-treated Lactobacillus spp. or their cell-free spent broth inhibited the growth of C. albicans and should be investigated for possible use in anti-Candida probiotic formulations in future.     

2-Amino-nonyl-6-methoxyl-tetralin muriate inhibits sterol C-14 reductase in the ergosterol biosynthetic pathway

Aim:

To investigate the action mechanism of a novel chemical structural aminotetralin derivate, 2-Amino-Nonyl-6-Methoxyl-Tetralin Muriate (10b), against Candida albicans (C albicans) in the ergosterol biosynthetic pathway.

Methods:

Antifungal susceptibility test of 10b was carried out using broth microdilution method, the action mechanism of 10b against C albicans was investigated by GC-MS spectrometry and real-time RT-PCR assay, and cytotoxicity of 10b in vitro was assessed by MTS/PMS reduction assay.

Results:

10b reduced the ergosterol content markedly, and the 50% ergosterol content inhibitory concentration (ECIC₅₀ value) was 0.08 μg/mL. Although the sterol composition of 10b-grown cells was completely identical with that of erg24 strain, the content of ergosta-8,14,22-trienol in 10b-grown cells was much higher than that in erg24 strain. Real-time RT-PCR assay revealed a global upregulation of sterol metabolism genes. In addition, the 50% inhibitory concentration (IC₅₀ value) of 10b was 11.30 μg/mL for murine embryonic fibroblasts and 35.70 μg/mL for human normal liver cells.

Conclusion:

10b possessed a mode of action different from that of azoles and morpholines, whose targets were sterol C-14 reductase (encoded by ERG24 gene) and sterol C-5 desaturase (encoded by ERG3) related enzyme. Although 10b seemed to reduce MTS/PMS reduction in a dose dependent manner, IC₅₀ value for mammalian cells was much higher than 50% minimum inhibitory concentration (MIC₅₀) value for C albicans. This indicates that the formulation is preliminarily safe and warrants further study for possible human applications.     

A novel polyamide SL-A92 as a potential fungal resistance blocker： synthesis and bioactivities in

Aim:

To synthesize a novel polyamide SL-A92 and evaluate its bioactivity against drug resistance in Candida albicans.

Methods:

SL-A92 was synthesized using N-hydroxybenzotriazole (HOBT)/N,N''-dicyclohexylcarbodiimide (DCC) in solution phase. Its antifungal activities and effects on strain growth were tested using the micro-broth dilution method and growth curves, respectively. Induced drug resistance in the C. albicans collection strain SC5314 was obtained by incubation with fluconazole (12 μg/mL) for 21 passages. Meanwhile, incubations with SL-A92 plus fluconazole were also carried out in SC5314 strains, and the MIC₈₀s were used to evaluate the inhibitory effects of SL-A92 on drug resistance during the induction process. Real time RT-PCR was performed to investigate the CDR1 and CDR2 mRNA levels in induced SC5314 strains.

Results:

SC5314 strain induced by the combination of fluconazole and SL-A92 (200 μg/mL) did not develop drug resistance. On day 24, the CDR1 and CDR2 mRNA levels in SC5314 strain co-treated with fluconazole and SL-A92 relative to fluconazole alone were 26% and 24%, respectively, and on day 30 the CDR1 and CDR2 mRNA levels were 43% and 31%, respectively.

Conclusion:

SL-A92 can block the development of drug resistance during the fluconazole induction process, which partially results from the down-regulation of CDR1 and CDR2.     

Fluopsin C induces oncosis of human breast adenocarcinoma cells

Aim:

Fluopsin C, an antibiotic isolated from Pseudomonas jinanesis, has shown antitumor effects on several cancer cell lines. In the current study, the oncotic cell death induced by fluopsin C was investigated in human breast adenocarcinoma cells in vitro.

Methods:

Human breast adenocarcinoma cell lines MCF-7 and MD-MBA-231 were used. The cytotoxicity was evaluated using MTT assay. Time-lapse microscopy and transmission electron microscopy were used to observe the morphological changes. Cell membrane integrity was assessed with propidium iodide (PI) uptake and lactate dehydrogenase (LDH) assay. Flow cytometry was used to measure reactive oxygen species (ROS) level and mitochondrial membrane potential (Δψm). A multimode microplate reader was used to analyze the intracellular ATP level. The changes in cytoskeletal system were investigated with Western blotting and immunostaining.

Results:

Fluopsin C (0.5-8 μmol/L) reduced the cell viability in dose- and time-dependent manners. Its IC₅₀ values in MCF-7 and MD-MBA-231 cells at 24 h were 0.9 and 1.03 μmol/L, respectively. Fluopsin C (2 μmol/L) induced oncosis in both the breast adenocarcinoma cells characterized by membrane blebbing and swelling, which was blocked by pretreatment with the pan-caspase inhibitor Z-VAD-fmk. In MCF-7 cells, fluopsin C caused PI uptake into the cells, significantly increased LDH release, induced cytoskeletal system degradation and ROS accumulation, decreased the intracellular ATP level and Δψm. Noticeably, fluopsin C exerted comparable cytotoxicity against the normal human hepatocytes (HL7702) and human mammary epithelial cells with the IC₅₀ values at 24 h of 2.7 and 2.4 μmol/L, respectively.

Conclusion:

Oncotic cell death was involved in the anticancer effects of fluopsin C on human breast adenocarcinoma cells in vitro. The hepatoxicity of fluopsin C should not be ignored.     

Pilot study of rosiglitazone as an in vivo probe of paclitaxel exposure

AIMS

To evaluate the use of rosiglitazone and the erythromycin breath test (ERMBT), as probes of CYP2C8 and CYP3A4, respectively, to explain inter-individual variability in paclitaxel exposure.

METHODS

The concentration of rosiglitazone at 3 h and ERMBT results were included in a regression model to explain the variability in paclitaxel exposure in 14 subjects.

RESULTS

Rosiglitazone concentration was significantly correlated with paclitaxel exposure (P = 0.018) while ERMBT had no predictive value (P = 0.47).

CONCLUSIONS

The correlation between the exposure of rosiglitazone and paclitaxel likely reflects mutual dependence on the activity of CYP2C8. Rosiglitazone or similar agents may have value as in vivo probes of CYP2C8 activity.     

Preliminary isolation and in vitro antiyeast activity of active fraction from crude extract of Gracilaria changii

Objective:

To isolate the active fraction from crude extract of Gracilaria changii and to determine its in vitro antifungal activity.

Materials and Methods:

The active fraction was isolated from the crude extract of G. changii by various purification procedures such as column chromatography, thin layer chromatography, bioauthograph etc. The in vitro antifungal activity (Candida albicans) of the active fraction (1.00, 0.50, and 0.25 mg/ml) was studied by disc diffusion method and the effect of the active fraction on the morphology of yeast was done by scanning electron microscope (SEM) studies.

Results:

An active fraction with remarkable antifungal activity was separated from the crude extract. The active fraction was effective as a fungicide against C. albicans and showed a dose-dependent antifungal activity. A Scanning Electron Microscope (SEM) study confirmed the fungicidal effect of G. changii active fraction on C. albicans, by changing the normal morphology of C. albicans.

Conclusion:

From G. changii crude extract, an active fraction with remarkable in vitro antifungal activity has been isolated.     

Uncoupling of sarcoplasmic reticulum Ca²⁺-ATPase by N-arachidonoyl dopamine. Members of the endocannabinoid family as thermogenic drugs

BACKGROUND AND PURPOSE

The sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) plays a role in thermogenesis. The exogenous compound capsaicin increased SERCA-mediated ATP hydrolysis not coupled to Ca²⁺ transport. Here, we have sought to identify endogenous compounds that may function as SERCA uncoupling agents.

EXPERIMENTAL APPROACH

Using isolated SR vesicles from rabbits, we have screened for endogenous compounds that uncouple SERCA. We have also studied their ability to deplete cytoplasmic ATP from human skeletal muscle cells in culture.

KEY RESULTS

Studies on SR vesicles showed that the endogenous lipid metabolite N-arachidonoyl dopamine (NADA) was a potent stimulator of SERCA uncoupling. NADA stabilized an E₁-like pump conformation that had a lower dephosphorylation rate, low affinity for Ca²⁺ at the luminal sites and a specific proteinase K cleavage pattern involving protection of the C-terminal p83C fragment from further cleavage. Moreover, we found a significantly decreased cytoplasmic ATP levels following treatment of skeletal muscle cells with 100 nM NADA. This effect was dependent on the presence of glucose and abolished by pretreatment with the specific SERCA inhibitor thapsigargin, regardless of the presence of glucose.

CONCLUSIONS AND IMPLICATIONS

NADA is an endogenous molecule that may function as SERCA uncoupling agent in vivo. Members of the endocannabinoid family exert concerted actions on several Ca²⁺-handling proteins. Uncoupling of SERCA by exogenous compounds could be a novel post-mitochondrial strategy for reduction of cellular ATP levels. In addition, signalling networks leading to SERCA uncoupling can be explored to study the importance of this ion pump in pathophysiological conditions related to metabolism.     

Transport and pharmacodynamics of albitiazolium, an antimalarial drug candidate

BACKGROUND AND PURPOSE

Choline analogues, a new type of antimalarials, exert potent in vitro and in vivo antimalarial activity. This has given rise to albitiazolium, which is currently in phase II clinical trials to cure severe malaria. Here we dissected its mechanism of action step by step from choline entry into the infected erythrocyte to its effect on phosphatidylcholine (PC) biosynthesis.

EXPERIMENTAL APPROACH

We biochemically unravelled the transport and enzymatic steps that mediate de novo synthesis of PC and elucidated how albitiazolium enters the intracellular parasites and affects the PC biosynthesis.

KEY RESULTS

Choline entry into Plasmodium falciparum-infected erythrocytes is achieved both by the remnant erythrocyte choline carrier and by parasite-induced new permeability pathways (NPP), while parasite entry involves a poly-specific cation transporter. Albitiazolium specifically prevented choline incorporation into its end-product PC, and its antimalarial activity was strongly antagonized by choline. Albitiazolium entered the infected erythrocyte mainly via a furosemide-sensitive NPP and was transported into the parasite by a poly-specific cation carrier. Albitiazolium competitively inhibited choline entry via the parasite-derived cation transporter and also, at a much higher concentration, affected each of the three enzymes conducting de novo synthesis of PC.

CONCLUSIONS AND IMPLICATIONS

Inhibition of choline entry into the parasite appears to be the primary mechanism by which albitiazolium exerts its potent antimalarial effect. However, the pharmacological response to albitiazolium involves molecular interactions with different steps of the de novo PC biosynthesis pathway, which would help to delay the development of resistance to this drug.     

Proteome mapping of overexpressed membrane-enriched and cytosolic proteins in sodium antimony gluconate (SAG) resistant clinical isolate of Leishmania donovani

AIMS

This study aimed to identify differentially overexpressed membrane-enriched as well as cytosolic proteins in SAG sensitive and resistant clinical strains of L. donovani isolated from VL patients which are involved in the drug resistance mechanism.

METHODS

The proteins in the membrane-enriched as well as cytosolic fractions of drug-sensitive as well as drug-resistant clinical isolates were separated using two-dimensional gel electrophoresis and overexpressed identified protein spots of interest were excised and analysed using MALDI-TOF/TOF.

RESULTS

Six out of 12 overexpressed proteins were identified in the membrane-enriched fraction of the SAG resistant strain of L. donovani whereas 14 out of 18 spots were identified in the cytosolic fraction as compared with the SAG sensitive strain. The major proteins in the membrane-enriched fraction were ABC transporter, HSP-83, GPI protein transamidase, cysteine–leucine rich protein and 60S ribosomal protein L23a whereas in the cytosolic fraction proliferative cell nuclear antigen (PCNA), proteasome alpha 5 subunit, carboxypeptidase, HSP-70, enolase, fructose-1,6-bisphosphate aldolase, tubulin-beta chain have been identified. Most of these proteins have been reported as potential drug targets, except 60S ribosomal protein L23a and PCNA which have not been reported to date for their possible involvement in drug resistance against VL.

CONCLUSION

This study for the first time provided a cumulative proteomic analysis of proteins overexpressed in drug resistant clinical isolates of L. donovani indicating their possible role in antimony resistance of the parasite. Identified proteins provide a vast field to be exploited for novel treatment strategies against VL such as cloning and overexpression of these targets to produce recombinant therapeutic/prophylactic proteins.     

Differential enhancement of leukaemia cell differentiation without elevation of intracellular calcium by plant-derived sesquiterpene lactone compounds

Background and purpose:

All-trans retinoic acid (ATRA) induces complete remission in a majority of acute promyelocytic leukaemia patients, but resistance of leukaemic cells to ATRA and its toxicity, such as hypercalcaemia, lead to a limitation of treatment. Therefore, combination therapies with differentiation-enhancing agents at non-toxic concentrations of ATRA may overcome its side effects. Here, we investigated the effect of plant-derived sesquiterpene lactone compounds and their underlying mechanisms in ATRA-induced differentiation of human leukaemia HL-60 cells.

Experimental approach:

HL-60 cells were treated with four sesquiterpene lactones (helenalin, costunolide, parthenolide and sclareolide) and cell differentiation was determined by NBT reduction, Giemsa and cytofluorometric analyses. Signalling pathways were assessed by western blotting, gel-shift assay and kinase activity determinations and intracellular calcium levels were determined using a calcium-specific fluorescent probe.

Key results:

Helenalin, costunolide and parthenolide, but not sclareolide, increased ATRA-induced HL-60 cell differentiation into a granulocytic lineage. Signalling kinases PKC and ERK were involved in the ATRA-induced differentiation enhanced by all of the effective sesquiterpene lactones, but JNK and PI3-K were involved in the ATRA-induced differentiation enhanced by costunolide and parthenolide. Enhancement of cell differentiation closely correlated with inhibition of NF-κB DNA-binding activity by all three effective compounds. Importantly, enhancement of differentiation induced by 50 nM ATRA by the sesquiterpene lactones was not accompanied by elevation of basal intracellular calcium concentrations.

Conclusions and implications:

These results indicate that plant-derived sesquiterpene lactones may enhance ATRA-mediated cell differentiation through distinct pathways.     

Insights into the preclinical treatment of blood-stage malaria by the antibiotic borrelidin

Background and Purpose

Blood-stage Plasmodium parasites cause morbidity and mortality from malaria. Parasite resistance to drugs makes development of new chemotherapies an urgency. Aminoacyl-tRNA synthetases have been validated as antimalarial drug targets. We explored long-term effects of borrelidin and mupirocin in lethal P. yoelii murine malaria.

Experimental Approach

Long-term (up to 340 days) immunological responses to borrelidin or mupirocin were measured after an initial 4 day suppressive test. Prophylaxis and cure were evaluated and the inhibitory effect on the parasites analysed.

Key Results

Borrelidin protected against lethal malaria at 0.25 mg·kg⁻¹·day⁻¹. Antimalarial activity of borrelidin correlated with accumulation of trophozoites in peripheral blood. All infected mice treated with borrelidin survived and subsequently developed immunity protecting them from re-infection on further challenges, 75 and 340 days after the initial infection. This long-term immunity in borrelidin-treated mice resulted in negligible parasitaemia after re-infections and marked increases in total serum levels of antiparasite IgGs with augmented avidity. Long-term memory IgGs mainly reacted against high and low molecular weight parasite antigens. Immunofluorescence microscopy showed that circulating IgGs bound predominantly to late intracellular stage parasites, mainly schizonts.

Conclusions and Implications

Low borrelidin doses protected mice from lethal malaria infections and induced protective immune responses after treatment. Development of combination therapies with borrelidin and selective modifications of the borrelidin molecule to specifically inhibit plasmodial threonyl tRNA synthetase should improve therapeutic strategies for malaria.     

The new antihypertensive drug iptakalim activates ATP-sensitive potassium channels in the endothelium of resistance blood vessels

Aim:

To investigate the mechanisms underlying the activation of ATP-sensitive potassium channels (K_ATP) by iptakalim in cultured rat mesenteric microvascular endothelial cells (MVECs).

Methods:

Whole-cell K_ATP currents were recorded in MVECs using automated patch clamp devices. Nucleotides (ATP, ADP and UDP) were added to the internal perfusion system, whereas other drugs were added to the cell suspension on NPC-1 borosilicate glass chips.

Results:

Application of iptakalim (10 and 100 μmol/L) significantly increased the whole-cell K_ATP currents, which were prevented by the specific K_ATP blocker glibenclamide (1.0 μmol/L). The opening of K_ATP channels by iptakalim depended upon the intracellular concentrations of ATP or NDPs: iptakalim activated K_ATP channels when the intracellular ATP or NDPs were at 100 or 1000 μmol/L, and was ineffective when the non-hydrolysable ATP analogue ATPγS (1000 μmol/L) was infused into the cells. In contrast, the K_ATP opener pinacidil activated K_ATP channels when the intracellular concentrations of ATP or NDPs ranged from 10 to 5000 μmol/L, and even ATPγS (1000 μmol/L) was infused into the cells.

Conclusion:

Iptakalim activates K_ATP channels in the endothelial cells of resistance blood vessels with a low metabolic status, and this activation is dependent on both ATP hydrolysis and ATP ligands.     

Targeting mitochondrial cardiolipin and the cytochrome c/cardiolipin complex to promote electron transport and optimize mitochondrial ATP synthesis

BACKGROUND AND PURPOSE

Cardiolipin plays an important role in mitochondrial respiration and cardiolipin peroxidation is associated with age-related diseases. Hydrophobic interactions between cytochrome c and cardiolipin converts cytochrome c from an electron carrier to a peroxidase. In addition to cardiolipin peroxidation, this impedes electron flux and inhibits mitochondrial ATP synthesis. SS-31 (D-Arg-dimethylTyr-Lys-Phe-NH₂) selectively binds to cardiolipin and inhibits cytochrome c peroxidase activity. Here, we examined whether SS-31 also protected the electron carrier function of cytochrome c.

EXPERIMENTAL APPROACH

Interactions of SS-31 with cardiolipin were studied using liposomes and bicelles containing phosphatidylcholine alone or with cardiolipin. Structural interactions were assessed by fluorescence spectroscopy, turbidity and nuclear magnetic resonance. Effects of cardiolipin on electron transfer kinetics of cytochrome c were determined by cytochrome c reduction in vitro and oxygen consumption using mitoplasts, frozen and fresh mitochondria.

KEY RESULTS

SS-31 interacted only with liposomes and bicelles containing cardiolipin in about 1:1 ratio. NMR studies demonstrated that the aromatic residues of SS-31 penetrated deep into cardiolipin-containing bilayers. SS-31 restored cytochrome c reduction and mitochondrial oxygen consumption in the presence of added cardiolipin. In fresh mitochondria, SS-31 increased state 3 respiration and efficiency of ATP synthesis.

CONCLUSIONS AND IMPLICATIONS

SS-31 selectively targeted cardiolipin and modulated its interaction with cytochrome c. SS-31 inhibited the cytochrome c/cardiolipin complex peroxidase activity while protecting its ability to serve as an electron carrier, thus optimizing mitochondrial electron transport and ATP synthesis. This novel class of cardiolipin therapeutics has the potential to restore mitochondrial bioenergetics for treatment of numerous age-related diseases.

LINKED ARTICLES

This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8     

Proteomic screening of molecular targets of crocin

Background

Traditional drug discovery approaches are mainly relied on the observed phenotypic changes following administration of a plant extract, drug candidate or natural product. Recently, target-based approaches are becoming more popular. The present study aimed to identify the cellular targets of crocin, the bioactive dietary carotenoid present in saffron, using an affinity-based method.

Methods

Heart, kidney and brain tissues of BALB/c mice were homogenized and extracted for the experiments. Target deconvolution was carried out by first passing cell lysate through an affinity column prepared by covalently attaching crocin to agarose beads. Isolated proteins were separated on a 2D gel, trypsinized in situ and identified by MALDI-TOF/TOF mass spectrometry. MASCOT search engine was used to analyze Mass Data.

Results

Part of proteome that physically interacts with crocin was found to consist of beta-actin-like protein 2, cytochrome b-c1 complex subunit 1, ATP synthase subunit beta, tubulin beta-3 chain, tubulin beta-6 chain, 14-3-3 protein beta/alpha, V-type proton ATPase catalytic subunitA, 60 kDa heat shock protein, creatine kinase b-type, peroxiredoxin-2, cytochrome b-c1 complex subunit 2, acetyl-coA acetyltransferase, cytochrome c1, proteasome subunit alpha type-6 and proteasome subunit alpha type-4.

Conclusion

The present findings revealed that crocin physically binds to a wide range of cellular proteins such as structural proteins, membrane transporters, and enzymes involved in ATP and redox homeostasis and signal transduction.     

A variation in NOS1AP gene is associated with repaglinide efficacy on insulin resistance in type 2 diabetes of Chinese

Aim:

To investigate a potential association between SNP rs10494366 in the neural nitric oxide synthase adaptor protein (NOS1AP) and efficacy of repaglinide (an insulin secretagogue) in newly diagnosed Shanghai Chinese type 2 diabetes patients.

Methods:

A total of 104 newly diagnosed type 2 diabetes patients (69 men, 35 women) were recruited and treated with repaglinide for 24 weeks. Anthropometric measurements, clinical laboratory tests were obtained at baseline and after 24-week treatment. Genotyping was performed by sequencing.

Results:

The baseline value of BMI, HOMA-IR, HOMA-B, and fasting insulin level were significantly different between GG, GT, and TT genotypes (P=0.024, 0.030, 0.005, and 0.007, respectively). Carriers of TT genotype were in significant insulin resistance at baseline. After 24-week repaglinide monotherapy, the Δ value of fasting insulin (P=0.019) and HOMA-IR (P=0.011) were significantly different. TT carriers had the least insulin resistance after treatment. The mixed model analysis showed that the variation had an interaction effect with repaglinide treatment only on HOMA-IR (P=0.013).

Conclusion:

A common variant in rs10494366 is associated with repaglinide monotherapy efficacy on insulin resistance in newly diagnosed Shanghai Chinese type 2 diabetes patients.     

A study on the extracts of Cuscuta reflexa Roxb. in treatment of cyclophosphamide induced alopecia

Background

Alopecia is a dermatological disorder with psychosocial implications on patients with hair loss. Hair loss is one of the most feared side effects of chemotherapy. Plants have been widely used for hair growth promotion since ancient times in Ayurveda, Chinese and Unani systems of medicine. The effect of extracts of Cuscuta reflexa Roxb. in testosterone induced alopecia was reported.

Objective

In the present study, the efficacies of the extracts of Cuscuta reflexa in promoting hair growth in cyclophosphamide-induced hair loss have been determined.

Materials and methods

The study was performed by treated with petroleum ether and ethanolic extract of Cuscuta reflexa at the dose 250 mg/kg in male swiss albino rats. Cyclophosphamide (125 mg/kg) was used to induce alopecia.

Results

Groups treated with extracts of plant showed hair regrowth. Histopathology and gross morphologic observations for hair regrowth at shaved sites revealed active follicular proliferation.

Conclusions

It concluded that extracts of Cuscuta reflexa shown to be capable of promoting follicular proliferation or preventing hair loss in cyclophosphamide-induced hair fall.     

16α-Hydroxycleroda-3,13 (14)Z-dien-15,16-olide from Polyalthia longifolia: a safe and orally active antileishmanial agent

Background and purpose:

New antileishmanials from natural products are urgently needed due to the emergence of drug resistance complicated by severe cytotoxic effects. 16α-Hydroxycleroda-3,13 (14)Z-dien-15,16-olide (Compound 1) from Polyalthia longifolia was found to be a potential antileishmanial and non-cytotoxic, as evidenced by long-term survival (>6 months) of treated animals. This prompted us to determine its target and, using molecular modelling, identify the interactions responsible for its specific antileishmanial activity.

Experimental approach:

In vitro activity of compound was assessed using intracellular transgenic green fluorescent protein-stably expressed Leishmania donovani parasites. In vivo activity and survival of animals post-treatment were evaluated in L. donovani-infected hamsters. Known property of clerodane diterpenes as potent human DNA topoisomerase inhibitors led us to evaluate the inhibition of recombinant L. donovani topoisomerase I using relaxation assay. Mode of cell death induced by Compound 1 was assessed by phosphotidylserine exposure post-treatment. Molecular modelling studies were conducted with DNA topoisomerase I to identify the binding interactions responsible for its activity.

Key results:

Bioassay-guided fractionation led to isolation of Compound 1 as a non-cytotoxic, orally active antileishmanial. Compound 1 inhibited recombinant DNA topoisomerase I which, ultimately, induced apoptosis. Molecular docking studies indicated that five strong hydrogen-bonding interactions and hydrophobic interactions of Compound 1 with L. donovani DNA-topoisomerase are responsible for its antileishmanial activity.

Conclusions and implications:

The data reveal Compound 1 is a potent and safe antileishmanial. The study further exploited the structural determinants responsible for its non-cytotoxic and potent activity, to raise the feasibility of specifically targeting the target enzyme responsible for its activity through rational drug design.     

Arctigenin alleviates ER stress via activating AMPK

Aim:

To investigate the protective effects of arctigenin (ATG), a phenylpropanoid dibenzylbutyrolactone lignan from Arctium lappa L (Compositae), against ER stress in vitro and the underlying mechanisms.

Methods:

A cell-based screening assay for ER stress regulators was established. Cell viability was measured using MTT assay. PCR and Western blotting were used to analyze gene and protein expression. Silencing of the CaMKKβ, LKB1, and AMPKα1 genes was achieved by RNA interference (RNAi). An ATP bioluminescent assay kit was employed to measure the intracellular ATP levels.

Results:

ATG (2.5, 5 and 10 μmol/L) inhibited cell death and unfolded protein response (UPR) in a concentration-dependent manner in cells treated with the ER stress inducer brefeldin A (100 nmol/L). ATG (1, 5 and 10 μmol/L) significantly attenuated protein synthesis in cells through inhibiting mTOR-p70S6K signaling and eEF2 activity, which were partially reversed by silencing AMPKα1 with RNAi. ATG (1-50 μmol/L) reduced intracellular ATP level and activated AMPK through inhibiting complex I-mediated respiration. Pretreatment of cells with the AMPK inhibitor compound C (25 μmol/L) rescued the inhibitory effects of ATG on ER stress. Furthermore, ATG (2.5 and 5 μmol/L) efficiently activated AMPK and reduced the ER stress and cell death induced by palmitate (2 mmol/L) in INS-1 β cells.

Conclusion:

ATG is an effective ER stress alleviator, which protects cells against ER stress through activating AMPK, thus attenuating protein translation and reducing ER load.     

Functional reconstitution of Haemonchus contortus acetylcholine receptors in Xenopus oocytes provides mechanistic insights into levamisole resistance

BACKGROUND AND PURPOSE

The cholinergic agonist levamisole is widely used to treat parasitic nematode infestations. This anthelmintic drug paralyses worms by activating a class of levamisole-sensitive acetylcholine receptors (L-AChRs) expressed in nematode muscle cells. However, levamisole efficacy has been compromised by the emergence of drug-resistant parasites, especially in gastrointestinal nematodes such as Haemonchus contortus. We report here the first functional reconstitution and pharmacological characterization of H. contortus L-AChRs in a heterologous expression system.

EXPERIMENTAL APPROACH

In the free-living nematode Caenorhabditis elegans, five AChR subunit and three ancillary protein genes are necessary in vivo and in vitro to synthesize L-AChRs. We have cloned the H. contortus orthologues of these genes and expressed them in Xenopus oocytes. We reconstituted two types of H. contortus L-AChRs with distinct pharmacologies by combining different receptor subunits.

KEY RESULTS

The Hco-ACR-8 subunit plays a pivotal role in selective sensitivity to levamisole. As observed with C. elegans L-AChRs, expression of H. contortus receptors requires the ancillary proteins Hco-RIC-3, Hco-UNC-50 and Hco-UNC-74. Using this experimental system, we demonstrated that a truncated Hco-UNC-63 L-AChR subunit, which was specifically detected in a levamisole-resistant H. contortus isolate, but not in levamisole-sensitive strains, hampers the normal function of L-AChRs, when co-expressed with its full-length counterpart.

CONCLUSIONS AND IMPLICATIONS

We provide the first functional evidence for a putative molecular mechanism involved in levamisole resistance in any parasitic nematode. This expression system will provide a means to analyse molecular polymorphisms associated with drug resistance at the electrophysiological level.     

WSS45, a sulfated a-D-glucan, strongly interferes with Dengue 2 virus infection in vitro

Aim:

To investigate the mode of action of WSS45, one sulfated derivative of an α-D-glucan from the Gastrodia elata Bl, on the multiplication cycle of dengue virus serotype 2 (DV2), including initial infection and intracellular replication.

Methods:

Virus multiplication in BHK cells were monitored by qRT-PCR, plaque reduction assay, and flow cytometry. Initial virus infection was dissected into adsorption and penetration steps by converting temperature and treating by acid glycine. Surface bound virions were detected by immunofluorescence staining for Evelope protein.

Results:

WSS45 effectively inhibited DV2 infection in BHK cells with an EC₅₀ value of 0.68±0.17 μg/mL , mainly interfered with virus adsorption, in a very early stage of the virus cycle. However, WSS45 showed no viricidal effect. Moreover, WSS45 could increase the detaching of virus from cell surface in BHK cell line.

Conclusion:

WSS45 exerted potent inhibitory effect on DV2 through interfering with the interaction between viruses and targeted cells. This activity was related to its molecular size.