Search

Background Treatment of Mycobacterium ulcerans disease, or Buruli ulcer (BU), has shifted from surgery to treatment with streptomycin(STR)+rifampin(RIF) since 2004 based on studies in a mouse model and clinical trials. We tested two entirely oral regimens for BU treatment, rifampin(RIF)+clarithromycin(CLR) and rifapentine(RPT)+clarithromycin(CLR) in the mouse model. Methodology/Principal Findings BALB/c mice were infected in the right hind footpad with M. ulcerans strain 1059 and treated daily (5 days/week) for 4 weeks, beginning 11 days after infection. Treatment groups included an untreated control, STR+RIF as a positive control, and test regimens of RIF, RPT, STR and CLR given alone and the RIF+CLR and RPT+CLR combinations. The relative efficacy of the drug treatments was compared on the basis of footpad CFU counts and median time to footpad swelling. Except for CLR, which was bacteriostatic, treatment with all other drugs reduced CFU counts by approximately 2 or 3 log10. Median time to footpad swelling after infection was 5.5, 16, 17, 23.5 and 36.5 weeks in mice receiving no treatment, CLR alone, RIF+CLR, RIF alone, and STR alone, respectively. At the end of follow-up, 39 weeks after infection, only 48%, 26.4% and 16.3% of mice treated with RPT+CLR, RPT alone and STR+RIF had developed swollen footpads. An in vitro checkerboard assay showed the interaction of CLR and RIF to be indifferent. However, in mice, co-administration with CLR resulted in a roughly 25% decrease in the maximal serum concentration (Cmax) and area under the serum concentration-time curve (AUC) of each rifamycin. Delaying the administration of CLR by one hour restored Cmax and AUC values of RIF to levels obtained with RIF alone. Conclusions/Significance These results suggest that an entirely oral daily regimen of RPT+CLR may be at least as effective as the currently recommended combination of injected STR+oral RIF., Author Summary Buruli ulcer (BU) is found throughout the world but is particularly prevalent in West Africa. Until 2004, treatment for this disfiguring disease was surgical excision followed by skin grafting, procedures often requiring months of hospitalization. More recently, an 8-week regimen of oral rifampin and streptomycin administered by injection has become the standard of care recommended by the World Health Organization. However, daily injections require sterile needles and syringes to prevent spread of blood borne pathogens and streptomycin has potentially serious side effects, most notably hearing loss. We tested an entirely oral regimen, substituting the long acting rifapentine for rifampin and clarithromycin for streptomycin. We also evaluated each drug separately. We found that rifapentine alone is as good as rifampin plus streptomycin, but the simultaneous addition of effective clarithromycin doses, at least in the mouse, reduces the activity of both rifampin and rifapentine, making it difficult to assess the efficacy of the oral regimens in the model. Studies of serum drug concentrations indicated that separating treatment times by one hour or reducing the clarithromycin dose to one active in humans should overcome this issue in experimental and clinical BU treatment, respectively.

BACKGROUND: Although human immunodeficiency virus (HIV)-related morbidity and mortality rates in patients treated with a combination of high active antiretroviral therapy (HAART) have declined, significant metabolic/vascular adverse effects associated with the long term use of HIV protease inhibitors (PIs) have emerged as a significant side effect. Here we illustrate that targeting the bile acid sensor farnesoid X receptor (FXR) protects against dyslipidemia and vascular injury induced HIV-PIs in rodents. METHODOLOGY/PRINCIPAL FINDINGS: Administration of the HIV PI ritonavir to wild type mice increased plasma triacylglycerols and cholesterol levels and this effect was exacerbated by dosing ritonavir to mice harbouring a disrupted FXR. Dyslipidemia induced by ritonavir associated with a shift in the liver expression of signature genes, Sterol Regulatory Element-Binding Protein (SREBP)-1 and fatty acid synthase. Treating wild type mice with the FXR agonist (chenodeoxycholic acid, CDCA) protected against development of dyslipidemia induced by ritonavir. Administration of ritonavir to ApoE(-/-) mice, a strain that develop spontaneously atherosclerosis, increased the extent of aortic plaques without worsening the dyslipidemia. Treating these mice with CDCA reduced the extent of aortic plaques by 70% without changing plasma lipoproteins or the liver expression of signature genes. A beneficial effect on aortic plaques was also obtained by treating ApoE(-/-) mice with gemfibrozil, a PPARα agonist. FXR activation counter-regulated induction of expression/activity of CD36 caused by HIV-PIs in circulating monocytes and aortic plaques. In macrophages cell lines, CDCA attenuated CD36 induction and uptake of acetylated LDL caused by ritonavir. Natural and synthetic FXR ligands reduced the nuclear translocation of SREBP1c caused by ritonavir. CONCLUSIONS/SIGNIFICANCE: Activation of the bile acid sensor FXR protects against dyslipidemia and atherosclerotic caused by ritonavir, a widely used HIV PI. From a mechanistic stand point it appears that besides reducing the liver expression of genes involved in fatty acid synthesis, FXR activation counter-regulates the expression/activity of CD36 on monocytes. FXR ligands might hold promise in the treatment dyslipidemia induced by ritonavir.

Background Long-term potentiation (LTP) at the parallel fibre–Purkinje cell synapse in the cerebellum is a recently described and poorly characterized form of synaptic plasticity. The induction mechanism for LTP at this synapse is considered reciprocal to “classical” LTP at hippocampal CA1 pyramidal neurons: kinases promote increased trafficking of AMPA receptors into the postsynaptic density in the hippocampus, whereas phosphatases decrease internalization of AMPA receptors in the cerebellum. In the hippocampus, LTP occurs in overlapping phases, with the transition from early to late phases requiring the consolidation of initial induction processes by structural re-arrangements at the synapse. Many signalling pathways have been implicated in this process, including PI3 kinases and Rho GTPases. Principal Findings We hypothesized that analogous phases are present in cerebellar LTP, and took as the starting point for investigation our recent discovery that P-Rex – a Rac guanine nucleotide exchange factor which is activated by PtdIns(3,4,5)P3 – is highly expressed in mouse cerebellar Purkinje neurons and plays a role in motor coordination. We found that LTP evoked at parallel fibre synapses by 1 Hz stimulation or by NO donors was not sustained beyond 30 min when P-Rex was eliminated or Rac inhibited, suggesting that cerebellar LTP exhibits a late phase analogous to hippocampal LTP. In contrast, inhibition of PI3 kinase activity eliminated LTP at the induction stage. Conclusions Our data suggest that a PI3K/P-Rex/Rac pathway is required for late phase LTP in the mouse cerebellum, and that other PI3K targets, which remain to be discovered, control LTP induction.

Predicting off-targets by computational methods is getting increasing importance in early drug discovery stages. We herewith present a computational method based on binding site three-dimensional comparisons, which prompted us to investigate the cross-reaction of protein kinase inhibitors with synapsin I, an ATP-binding protein regulating neurotransmitter release in the synapse. Systematic pair-wise comparison of the staurosporine-binding site of the proto-oncogene Pim-1 kinase with 6,412 druggable protein-ligand binding sites suggested that the ATP-binding site of synapsin I may recognize the pan-kinase inhibitor staurosporine. Biochemical validation of this hypothesis was realized by competition experiments of staurosporine with ATP-gamma(35)S for binding to synapsin I. Staurosporine, as well as three other inhibitors of protein kinases (cdk2, Pim-1 and casein kinase type 2), effectively bound to synapsin I with nanomolar affinities and promoted synapsin-induced F-actin bundling. The selective Pim-1 kinase inhibitor quercetagetin was shown to be the most potent synapsin I binder (IC50 = 0.15 microM), in agreement with the predicted binding site similarities between synapsin I and various protein kinases. Other protein kinase inhibitors (protein kinase A and chk1 inhibitor), kinase inhibitors (diacylglycerolkinase inhibitor) and various other ATP-competitors (DNA topoisomerase II and HSP-90alpha inhibitors) did not bind to synapsin I, as predicted from a lower similarity of their respective ATP-binding sites to that of synapsin I. The present data suggest that the observed downregulation of neurotransmitter release by some but not all protein kinase inhibitors may also be contributed by a direct binding to synapsin I and phosphorylation-independent perturbation of synapsin I function. More generally, the data also demonstrate that cross-reactivity with various targets may be detected by systematic pair-wise similarity measurement of ligand-annotated binding sites.

International audience; BACKGROUND: 6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the identification of cellular targets of these drugs. Using affinity chromatography matrices for both drugs, we demonstrate an RNA-dependent interaction of 6AP and GA with the ribosome. These specific interactions have no effect on the peptidyl transferase activity of the ribosome or on global translation. In contrast, 6AP and GA specifically inhibit the ribosomal RNA-mediated protein folding activity of the ribosome. CONCLUSION/SIGNIFICANCE: 6AP and GA are therefore the first compounds to selectively inhibit the protein folding activity of the ribosome. They thus constitute precious tools to study the yet largely unexplored biological role of this protein folding activity.

Background Severe Combined Immune Deficient (SCID)/Urokinase-type Plasminogen Activator (uPA) mice undergo liver failure and are useful hosts for the propagation of transplanted human hepatocytes (HH) which must compete with recipient-derived hepatocytes for replacement of the diseased liver parenchyma. While partial replacement by HH has proven useful for studies with Hepatitis C virus, complete replacement of SCID/uPA mouse liver by HH has never been achieved and limits the broader application of these mice for other areas of biomedical research. The herpes simplex virus type-1 thymidine kinase (HSVtk)/ganciclovir (GCV) system is a powerful tool for cell-specific ablation in transgenic animals. The aim of this study was to selectively eliminate murine-derived parenchymal liver cells from humanized SCID/uPA mouse liver in order to achieve mice with completely humanized liver parenchyma. Thus, we reproduced the HSVtk (vTK)/GCV system of hepatic failure in SCID/uPA mice. Methodology/Principal Findings In vitro experiments demonstrated efficient killing of vTK expressing hepatoma cells after GCV treatment. For in vivo experiments, expression of vTK was targeted to the livers of FVB/N and SCID/uPA mice. Hepatic sensitivity to GCV was first established in FVB/N mice since these mice do not undergo liver failure inherent to SCID/uPA mice. Hepatic vTK expression was found to be an integral component of GCV-induced pathologic and biochemical alterations and caused death due to liver dysfunction in vTK transgenic FVB/N and non-transplanted SCID/uPA mice. In SCID/uPA mice with humanized liver, vTK/GCV caused death despite extensive replacement of the mouse liver parenchyma with HH (ranging from 32–87%). Surprisingly, vTK/GCV-dependent apoptosis and mitochondrial aberrations were also localized to bystander vTK-negative HH. Conclusions/Significance Extensive replacement of mouse liver parenchyma by HH does not provide a secure therapeutic advantage against vTK/GCV-induced cytotoxicity targeted to residual mouse hepatocytes. Functional support by engrafted HH may be secured by strategies aimed at limiting this bystander effect.

Background Cystic Echinococosis (CE) is a zoonotic disease caused by larval stage Echinococcus granulosus. We determined the effects of high dose of Oxfendazole (OXF), combination Oxfendazole/Praziquantel (PZQ), and combination Albendazole (ABZ)/Praziquantel against CE in sheep. Methodology/Principal Findings A randomized placebo-controlled trial was carried out on 118 randomly selected ewes. They were randomly assigned to one of the following groups: 1) placebo; 2) OXF 60 mg/Kg of body weight (BW) weekly for four weeks; 3) ABZ 30 mg/Kg BW + PZQ 40 mg/Kg BW weekly for 6 weeks, and 4) OXF 30 mg/Kg BW+ PZQ 40 mg/Kg BW biweekly for 3 administrations (6 weeks). Percent protoscolex (PSC) viability was evaluated using a 0.1% aqueous eosin vital stain for each cyst. “Noninfective” sheep were those that had no viable PSCs; “low-medium infective” were those that had 1% to 60% PSC viability; and “high infective” were those with more than 60% PSC viability. We evaluated 92 of the 118 sheep. ABZ/PZQ led the lowest PSC viability for lung cysts (12.7%), while OXF/PZQ did so for liver cysts (13.5%). The percentage of either “noninfective” or “low-medium infective” sheep was 90%, 93.8% and 88.9% for OXF, ABZ/PZQ and OXF/PZQ group as compared to 50% “noninfective” or “low-medium infective” for placebo. After performing all necropsies, CE prevalence in the flock of sheep was 95.7% (88/92) with a total number of 1094 cysts (12.4 cysts/animal). On average, the two-drug-combination groups resulted pulmonary cysts that were 6 mm smaller and hepatic cysts that were 4.2 mm smaller than placebo (p, Author Summary Cystic Echinococcosis (CE) is a near-cosmopolitan parasitic zoonosis that causes economic losses in many regions of the world. This parasitic infection can be regarded as an emerging or re-emerging disease causing considerable losses in livestock production. CE is produced by the larval cystic stage (hydatid) of the dog parasite Echinococcus granulosus. After infective eggs are ingested, cysts develop mainly in lungs and liver of humans and animals (sheep, cattle, pigs, horses, etc). Infected people may require surgery and/or Albendazole-based chemotherapy. In this study, we evaluated the effects of Oxfendazole alone (an antiparasitic drug used in animals), Oxfendazole plus Praziquantel, and Albendazole plus Praziquantel against hydatid cysts in sheep over 4 to 6 weeks of treatment. All of the treatments in this study were efficacious in killing the larval stages and, therefore, in minimizing the risk of a dog acquiring new infections (taenias). These treatment schemes can be added to control measures in animals and eventually could be used for the treatment of human infection. Further investigations on different schedules of monotherapy or combined chemotherapy are needed, as well as studies to evaluate the safety and efficacy of Oxfendazole in humans.

Adverse drug reactions (ADR), also known as side-effects, are complex undesired physiologic phenomena observed secondary to the administration of pharmaceuticals. Several phenomena underlie the emergence of each ADR; however, a dominant factor is the drug's ability to modulate one or more biological pathways. Understanding the biological processes behind the occurrence of ADRs would lead to the development of safer and more effective drugs. At present, no method exists to discover these ADR-pathway associations. In this paper we introduce a computational framework for identifying a subset of these associations based on the assumption that drugs capable of modulating the same pathway may induce similar ADRs. Our model exploits multiple information resources. First, we utilize a publicly available dataset pairing drugs with their observed ADRs. Second, we identify putative protein targets for each drug using the protein structure database and in-silico virtual docking. Third, we label each protein target with its known involvement in one or more biological pathways. Finally, the relationships among these information sources are mined using multiple stages of logistic-regression while controlling for over-fitting and multiple-hypothesis testing. As proof-of-concept, we examined a dataset of 506 ADRs, 730 drugs, and 830 human protein targets. Our method yielded 185 ADR-pathway associations of which 45 were selected to undergo a manual literature review. We found 32 associations to be supported by the scientific literature.

BackgroundMost medicinal plants contain a mixture of bioactive compounds, including chemicals that interact with intracellular targets and others that can act as adjuvants to facilitate absorption of polar agents across cellular membranes. However, little is known about synergistic effects between such potential drug candidates and adjuvants. To probe for such effects, we tested the green tea compound epigallocatechin gallate (EGCG) and the membrane permeabilising digitonin on Plasmodium sporozoite motility and viability.Methodology/principal findingsGreen fluorescent P. berghei sporozoites were imaged using a recently developed visual screening methodology. Motility and viability parameters were automatically analyzed and IC50 values were calculated, and the synergism of drug and adjuvant was assessed by the fractional inhibitory concentration index. Validating our visual screening procedure, we showed that sporozoite motility and liver cell infection is inhibited by EGCG at nontoxic concentrations. Digitonin synergistically increases the cytotoxicity of EGCG on sporozoite survival, but shows an additive effect on sporozoite motility.Conclusions/significanceWe proved the feasibility of performing highly reliable visual screens for compounds against Plasmodium sporozoites. We thereby could show an advantage of administering mixtures of plant metabolites on inhibition of cell motility and survival. Although the effective concentration of both drugs is too high for use in malaria prophylaxis, the demonstration of a synergistic effect between two plant compounds could lead to new avenues in drug discovery.

Understanding the mechanisms of cell function and drug action is a major endeavor in the pharmaceutical industry. Drug effects are governed by the intrinsic properties of the drug (i.e., selectivity and potency) and the specific signaling transduction network of the host (i.e., normal vs. diseased cells). Here, we describe an unbiased, phosphoproteomic-based approach to identify drug effects by monitoring drug-induced topology alterations. With our proposed method, drug effects are investigated under diverse stimulations of the signaling network. Starting with a generic pathway made of logical gates, we build a cell-type specific map by constraining it to fit 13 key phopshoprotein signals under 55 experimental conditions. Fitting is performed via an Integer Linear Program (ILP) formulation and solution by standard ILP solvers; a procedure that drastically outperforms previous fitting schemes. Then, knowing the cell's topology, we monitor the same key phosphoprotein signals under the presence of drug and we re-optimize the specific map to reveal drug-induced topology alterations. To prove our case, we make a topology for the hepatocytic cell-line HepG2 and we evaluate the effects of 4 drugs: 3 selective inhibitors for the Epidermal Growth Factor Receptor (EGFR) and a non-selective drug. We confirm effects easily predictable from the drugs' main target (i.e., EGFR inhibitors blocks the EGFR pathway) but we also uncover unanticipated effects due to either drug promiscuity or the cell's specific topology. An interesting finding is that the selective EGFR inhibitor Gefitinib inhibits signaling downstream the Interleukin-1alpha (IL1α) pathway; an effect that cannot be extracted from binding affinity-based approaches. Our method represents an unbiased approach to identify drug effects on small to medium size pathways which is scalable to larger topologies with any type of signaling interventions (small molecules, RNAi, etc). The method can reveal drug effects on pathways, the cornerstone for identifying mechanisms of drug's efficacy., Author Summary Cells are complex functional units. Signal transduction refers to the underlying mechanism that regulates cell function, and it is usually depicted on signaling pathways maps. Each cell type has distinct signaling transduction mechanisms, and several diseases arise from alterations on the signaling pathways. Small-molecule inhibitors have emerged as novel pharmaceutical interventions that aim to block certain pathways in an effort to reverse the abnormal phenotype of the diseased cells. Despite that compounds have been well designed to hit certain molecules (i.e., targets), little is known on how they act on an “operative” signaling network. Here, we combine novel high throughput protein-signaling measurements and sophisticated computational techniques to evaluate drug effects on cells. Our approach comprises of two steps: build pathways that simulate cell function and identify drug-induced alterations of those pathways. We employed our approach to evaluate the effects of 4 drugs on a cancer hepatocytic cell type. We were able to confirm the main target of the drugs but also uncover unknown off-target effects. By understanding the drug effects in normal and diseased cells we can provide important information for the analysis of clinical outcomes in order to improve drug efficacy and safety.

Background Nuclear Magnetic Resonance (NMR) spectroscopy offers a variety of experiments to study protein-ligand interactions at atomic resolution. Among these experiments, 15N Heteronuclear Single Quantum Correlation (HSQC)experiment is simple, less time consuming and highly informative in mapping the binding site of the ligand. The interpretation of 15N HSQC becomes ambiguous when the chemical shift perturbations are caused by non-specific interactions like allosteric changes and local structural rearrangement. Under such cases, detailed chemical exchange analysis based on chemical shift perturbation will assist in locating the binding site accurately. Methodology/principal findings We have automated the mapping of binding sites for fast chemical exchange systems using information obtained from 15N HSQC spectra of protein serially titrated with ligand of increasing concentrations. The automated program Auto-FACE (Auto-FAst Chemical Exchange analyzer) determines the parameters, e.g. rate of change of perturbation, binding equilibrium constant and magnitude of chemical shift perturbation to map the binding site residues.Interestingly, the rate of change of perturbation at lower ligand concentration is highly sensitive in differentiating the binding site residues from the non-binding site residues. To validate this program, the interaction between the protein hBcl(XL) and the ligand BH3I-1 was studied. Residues in the hydrophobic BH3 binding groove of hBcl(XL) were easily identified to be crucial for interaction with BH3I-1 from other residues that also exhibited perturbation. The geometrically averaged equilibrium constant (3.0 x 10(4)) calculated for the residues present at the identified binding site is consistent with the values obtained by other techniques like isothermal calorimetry and fluorescence polarization assays (12.8 x 10(4)). Adjacent to the primary site, an additional binding site was identified which had an affinity of 3.8 times weaker than the former one. Further NMR based model fitting for individual residues suggest single site model for residues present at these binding sites and two site model for residues present between these sites. This implies that chemical shift perturbation can represent the local binding event much more accurately than the global binding event. Conclusion/significance Detail NMR chemical shift perturbation analysis enabled binding site residues to be distinguished from non-binding site residues for accurate mapping of interaction site in complex fast exchange system between small molecule and protein. The methodology is automated and implemented in a program called "Auto-FACE", which also allowed quantitative information of each interaction site and elucidation of binding mechanism.

BACKGROUND: The psychotomimetics ketamine and MK-801, non-competitive NMDA receptor (NMDAr) antagonists, induce cognitive impairment and aggravate schizophrenia symptoms. In conscious rats, they produce an abnormal behavior associated with a peculiar brain state characterized by increased synchronization in ongoing gamma (30-80 Hz) oscillations in the frontoparietal (sensorimotor) electrocorticogram (ECoG). This study investigated whether NMDAr antagonists-induced aberrant gamma oscillations are correlated with locomotion and dependent on hyperlocomotion-related sensorimotor processing. This also implied to explore the contribution of intracortical and subcortical networks in the generation of these pathophysiological ECoG gamma oscillations. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative locomotion data collected with a computer-assisted video tracking system in combination with ECoG revealed that ketamine and MK-801 induce highly correlated hyperlocomotion and aberrant gamma oscillations. This abnormal gamma hyperactivity was recorded over the frontal, parietal and occipital cortices. ECoG conducted under diverse consciousness states (with diverse anesthetics) revealed that NMDAr antagonists dramatically increase the power of basal gamma oscillations. Paired ECoG and intracortical local field potential recordings showed that the ECoG mainly reflects gamma oscillations recorded in underlying intracortical networks. In addition, multisite recordings revealed that NMDAr antagonists dramatically enhance the amount of ongoing gamma oscillations in multiple cortical and subcortical structures, including the prefrontal cortex, accumbens, amygdala, basalis, hippocampus, striatum and thalamus. CONCLUSIONS/SIGNIFICANCE: NMDAr antagonists acutely produces, in the rodent CNS, generalized aberrant gamma oscillations, which are not dependent on hyperlocomotion-related brain state or conscious sensorimotor processing. These findings suggest that NMDAr hypofunction-related generalized gamma hypersynchronies represent an aberrant diffuse network noise, a potential electrophysiological correlate of a psychotic-like state. Such generalized noise might cause dysfunction of brain operations, including the impairments in cognition and sensorimotor integration seen in schizophrenia.

International audience; The amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) reverses dopamine and serotonin transporters to produce efflux of dopamine and serotonin, respectively, in regions of the brain that have been implicated in reward. However, the role of serotonin/dopamine interactions in the behavioral effects of MDMA remains unclear. We previously showed that MDMA-induced locomotion, serotonin and dopamine release are 5-HT(2B) receptor-dependent. The aim of the present study was to determine the contribution of serotonin and 5-HT(2B) receptors to the reinforcing properties of MDMA.We show here that 5-HT(2B) (-/-) mice do not exhibit behavioral sensitization or conditioned place preference following MDMA (10 mg/kg) injections. In addition, MDMA-induced reinstatement of conditioned place preference after extinction and locomotor sensitization development are each abolished by a 5-HT(2B) receptor antagonist (RS127445) in wild type mice. Accordingly, MDMA-induced dopamine D1 receptor-dependent phosphorylation of extracellular regulated kinase in nucleus accumbens is abolished in mice lacking functional 5-HT(2B) receptors. Nevertheless, high doses (30 mg/kg) of MDMA induce dopamine-dependent but serotonin and 5-HT(2B) receptor-independent behavioral effects.These results underpin the importance of 5-HT(2B) receptors in the reinforcing properties of MDMA and illustrate the importance of dose-dependent effects of MDMA on serotonin/dopamine interactions.

The widely used atypical antipsychotic clozapine is a potent competitive antagonist at 5-HT(3) receptors which may contribute to its unique psychopharmacological profile. Clozapine binds to 5-HT(3) receptors of various species. However, the structural requirements of the respective binding site for clozapine remain to be determined. Differences in the primary sequences within the 5-HT(3A) receptor gene in schizophrenic patients may result in an alteration of the antipsychotic potency and/or the side effect profile of clozapine. To determine these structural requirements we constructed chimeras with different 5-HT(3A) receptor sequences of murine and human origin and expressed these mutants in human embryonic kidney (HEK) 293 cells. Clozapine antagonises recombinant mouse 5-HT(3A) receptors with higher potency compared to recombinant human 5-HT(3A) receptors. 5-HT activation curves and clozapine inhibition curves yielded the parameters EC(50) and IC(50) for all receptors tested in the range of 0.6 - 2.7 microM and 1.5 - 83.3 nM, respectively. The use of the Cheng-Prusoff equation to calculate the dissociation constant K(b) values for clozapine revealed that an extracellular sequence (length 86 aa) close to the transmembrane domain M1 strongly determines the binding affinity of clozapine. K(b) values of clozapine were significantly lower (0.3-1.1 nM) for receptors containing the murine sequence and higher when compared with receptors containing the respective human sequence (5.8-13.4 nM). Thus, individual differences in the primary sequence of 5-HT(3) receptors may be crucial for the antipsychotic potency and/or the side effect profile of clozapine.

Background Alcoholism is a widespread chronic disorder of complex aetiology with a significant negative impact on the individual and the society. Mechanisms of ethanol action are not sufficiently well understood at the molecular level and the pharmacotherapy of alcoholism is still in its infancy. Our study focuses at the cellular and molecular level on ethanol-induced effects that are mediated through the mu-opioid receptor (MOP) and on the effects of naltrexone, a well-known antagonist at MOP that is used clinically to prevent relapse in alcoholism. Methodology/Principal Findings Advanced fluorescence imaging by Confocal Laser Scanning Microscopy (CLSM) and Fluorescence Correlation Spectroscopy (FCS) are used to study ethanol effects on MOP and plasma membrane lipid dynamics in live PC12 cells. We observed that relevant concentrations of ethanol (10–40 mM) alter MOP mobility and surface density, and affect the dynamics of plasma membrane lipids. Compared to the action of specific ligands at MOP, ethanol-induced effects show complex kinetics and point to a biphasic underlying mechanism. Pretreatment with naloxone or naltrexone considerably mitigates the effects of ethanol. Conclusions/Significance We suggest that ethanol acts by affecting the sorting of MOP at the plasma membrane of PC12 cells. Naltrexone exerts opposite effects on MOP sorting at the plasma membrane, thereby countering the effects of ethanol. Our experimental findings give new insight on MOP-mediated ethanol action at the cellular and molecular level. We suggest a new hypothesis to explain the well established ethanol-induced increase in the activity of the endogenous opioid system.

Astroglia are key cellular sites where opiate drug signals converge with the proinflammatory effects of HIV-1 Tat signals to exacerbate HIV encephalitis. Despite this understanding, the molecular sites of convergence driving opiate-accelerated neuropathogenesis have not been deciphered. We therefore explored potential points of interaction between the signaling pathways initiated by HIV-1 Tat and opioids in striatal astrocytes. Profiling studies screening 152 transcription factors indicated that the nuclear factor-kappa B (NF-kappaB) subunit, c-Rel, was a likely candidate for Tat or Tat plus opiate-induced increases in cytokine and chemokine production by astrocytes. Pretreatment with the NF-kappaB inhibitor parthenolide provided evidence that Tat+/-morphine-induced release of MCP-1, IL-6 and TNF-alpha by astrocytes is NF-kappaB dependent. The nuclear export inhibitor, leptomycin B, blocked the nucleocytoplasmic shuttling of NF-kappaB; causing p65 (RelA) accumulation in the nucleus, and significantly attenuated cytokine production in Tat+/-morphine exposed astrocytes. Similarly, chelating intracellular calcium ([Ca(2+)](i)) blocked Tat+/-morphine-evoked MCP-1 and IL-6 release, while artificially increasing the concentration of extracellular Ca(2+) reversed this effect. Taken together, these results demonstrate that: 1) exposure to Tat+/-morphine is sufficient to activate NF-kappaB and cytokine production, 2) the release of MCP-1 and IL-6 by Tat+/-morphine are highly Ca(2+)-dependent, while TNF-alpha appears to be less affected by the changes in [Ca(2+)](i), and 3) in the presence of Tat, exposure to opiates augments Tat-induced NF-kappaB activation and cytokine release through a Ca(2+)-dependent pathway.

To counter the global threat caused by Plasmodium falciparum malaria, new drugs and vaccines are urgently needed. However, there are no practical animal models because P. falciparum infects human erythrocytes almost exclusively. Here we describe a reliable falciparum murine model of malaria by generating strains of P. falciparum in vivo that can infect immunodeficient mice engrafted with human erythrocytes. We infected NOD(scid/beta2m-/-) mice engrafted with human erythrocytes with P. falciparum obtained from in vitro cultures. After apparent clearance, we obtained isolates of P. falciparum able to grow in peripheral blood of engrafted NOD(scid/beta2m-/-) mice. Of the isolates obtained, we expanded in vivo and established the isolate Pf3D7(0087/N9) as a reference strain for model development. Pf3D7(0087/N9) caused productive persistent infections in 100% of engrafted mice infected intravenously. The infection caused a relative anemia due to selective elimination of human erythrocytes by a mechanism dependent on parasite density in peripheral blood. Using this model, we implemented and validated a reproducible assay of antimalarial activity useful for drug discovery. Thus, our results demonstrate that P. falciparum contains clones able to grow reproducibly in mice engrafted with human erythrocytes without the use of myeloablative methods.

BackgroundType 2 diabetes often displays hyperlipidemia. We examined palmitate effects on pancreatic islet function in relation to FFA receptor GPR40, NO generation, insulin release, and the PPARgamma agonistic thiazolidinedione, rosiglitazone.Principal findingsRosiglitazone suppressed acute palmitate-stimulated GPR40-transduced PI hydrolysis in HEK293 cells and insulin release from MIN6c cells and mouse islets. Culturing islets 24 h with palmitate at 5 mmol/l glucose induced beta-cell iNOS expression as revealed by confocal microscopy and increased the activities of ncNOS and iNOS associated with suppression of glucose-stimulated insulin response. Rosiglitazone reversed these effects. The expression of iNOS after high-glucose culturing was unaffected by rosiglitazone. Downregulation of GPR40 by antisense treatment abrogated GPR40 expression and suppressed palmitate-induced iNOS activity and insulin release.ConclusionWe conclude that, in addition to mediating acute FFA-stimulated insulin release, GPR40 is an important regulator of iNOS expression and dysfunctional insulin release during long-term exposure to FFA. The adverse effects of palmitate were counteracted by rosiglitazone at GPR40, suggesting that thiazolidinediones are beneficial for beta-cell function in hyperlipidemic type 2 diabetes.

Background A recent drug interaction study reported that when azithromycin was administered with the combination of ivermectin and albendazole, there were modest increases in ivermectin pharmacokinetic parameters. Data from this study were reanalyzed to further explore this observation. A compartmental model was developed and 1,000 interaction studies were simulated to explore extreme high ivermectin values that might occur. Methods and Findings A two-compartment pharmacokinetic model with first-order elimination and absorption was developed. The chosen final model had 7 fixed-effect parameters and 8 random-effect parameters. Because some of the modeling parameters and their variances were not distributed normally, a second mixture model was developed to further explore these data. The mixture model had two additional fixed parameters and identified two populations, A (55% of subjects), where there was no change in bioavailability, and B (45% of subjects), where ivermectin bioavailability was increased 37%. Simulations of the data using both models were similar, and showed that the highest ivermectin concentrations fell in the range of 115–201 ng/mL. Conclusions This is the first pharmacokinetic model of ivermectin. It demonstrates the utility of two modeling approaches to explore drug interactions, especially where there may be population heterogeneity. The mechanism for the interaction was identified (an increase in bioavailability in one subpopulation). Simulations show that the maximum ivermectin exposures that might be observed during co-administration with azithromycin are below those previously shown to be safe and well tolerated. These analyses support further study of co-administration of azithromycin with the widely used agents ivermectin and albendazole, under field conditions in disease control programs., Author Summary This paper describes the use of a modeling and simulation approach to explore a reported pharmacokinetic interaction between two drugs (ivermectin and azithromycin), which along with albendazole, are being developed for combination use in neglected tropical diseases. This approach is complementary to more traditional pharmacokinetic and safety studies that need to be conducted to support combined use of different health interventions. A mathematical model of ivermectin pharmacokinetics was created and used to simulate multiple trials, and the probability of certain outcomes (very high peak blood ivermectin levels when given in combination) was determined. All simulated peak blood levels were within ranges known to be safe and well tolerated. Additional field studies are needed to confirm these findings.

Background With the phasing out of ozone-depleting substances in accordance with the United Nations Montreal Protocol, phosphine remains as the only economically viable fumigant for widespread use. However the development of high-level resistance in several pest insects threatens the future usage of phosphine; yet research into phosphine resistance mechanisms has been limited due to the potential for human poisoning in enclosed laboratory environments. Principal Findings Here we describe a custom-designed chamber for safely containing phosphine gas generated from aluminium phosphide tablets. In an improvement on previous generation systems, this chamber can be completely sealed to control the escape of phosphine. The device has been utilised in a screening program with C. elegans that has identified a phosphine synergist, and quantified the efficacy of a new fumigant against that of phosphine. The phosphine-induced mortality at 20°C has been determined with an LC50 of 732 ppm. This result was contrasted with the efficacy of a potential new botanical pesticide dimethyl disulphide, which for a 24 hour exposure at 20°C is 600 times more potent than phosphine (LC50 1.24 ppm). We also found that co-administration of the glutathione depletor diethyl maleate (DEM) with a sublethal dose of phosphine (70 ppm

Background The level of expression of functional α7-containing nicotinic acetylcholine receptors (nAChRs) in hippocampal CA1 pyramidal neurons is believed to be very low compared to hippocampal CA1 interneurons, and for many years this expression was largely overlooked. However, high densities of expression of functional α7-containing nAChRs in CA1 pyramidal neurons may not be necessary for triggering important cellular and network functions, especially if activation of α7-containing nAChRs occurs in the presence of positive allosteric modulators such as PNU-120596. Methodology/Principal Findings An approach previously developed for α7-containing nAChRs expressed in tuberomammillary neurons was applied to investigate functional CA1 pyramidal α7-containing nAChRs using rat coronal hippocampal slices and patch-clamp electrophysiology. The majority (∼71%) of tested CA1 pyramidal neurons expressed low densities of functional α7-containing nAChRs as evidenced by small whole-cell responses to choline, a selective endogenous agonist of α7 nAChRs. These responses were potentiated by PNU-120596, a novel positive allosteric modulator of α7 nAChRs. The density of functional α7-containing nAChRs expressed in CA1 pyramidal neurons (and thus, the normalized net effect of activation, i.e., response net charge per unit of membrane capacitance per unit of time) was estimated to be ∼5% of the density observed in CA1 interneurons. The results of this study demonstrate that despite low levels of expression of functional pyramidal α7-containing nAChRs, physiological levels of choline (∼10 µM) are sufficient to activate these receptors and transiently depolarize and even excite CA1 pyramidal neurons in the presence of PNU-120596. The observed effects are possible because in the presence of 10 µM choline and 1–5 µM PNU-120596, a single opening of an individual pyramidal α7-containing nAChR ion channel appears to transiently depolarize (∼4 mV) the entire pyramidal neuron and occasionally trigger action potentials. Conclusions 1) The majority of hippocampal CA1 pyramidal neurons express functional α7-containing nAChRs. In the absence of PNU-120596, a positive allosteric modulator of α7 nAChRs, a lack of responsiveness of some hippocampal CA1 pyramidal neurons to focal application of 0.5–1 mM choline does not imply a lack of expression of functional α7-containing nAChRs in these neurons. Rather, it may indicate a lack of detection of α7-containing nAChR-mediated currents by patch-clamp electrophysiology. 2) PNU-120596 can serve as a powerful tool for detection and enhancement of responsiveness of low densities of functional α7-containing nAChRs such as those present in hippocampal CA1 pyramidal neurons. 3) In the presence of PNU-120596, physiological concentrations of choline activate functional CA1 pyramidal α7-containing nAChRs and produce step-like currents that cause repetitive step-like depolarizations, occasionally triggering bursts of action potentials in CA1 pyramidal neurons. Therefore, the results of this study suggest that in the presence of PNU-120596 and possibly other positive allosteric modulators, endogenous choline may persistently activate CA1 pyramidal α7-containing nAChRs, enhance the excitability of CA1 pyramidal neurons and thus act as a potent therapeutic agent with potential neuroprotective and cognition-enhancing properties.

Background: Anecdotal information and case reports suggest that intravenously administered vitamin C is used by Complementary and Alternate Medicine (CAM) practitioners. The scale of such use in the U.S. and associated side effects are unknown. Methods and Findings: We surveyed attendees at annual CAM Conferences in 2006 and 2008, and determined sales of intravenous vitamin C by major U.S. manufacturers/distributors. We also queried practitioners for side effects, compiled published cases, and analyzed FDA’s Adverse Events Database. Of 199 survey respondents (out of 550), 172 practitioners administered IV vitamin C to 11,233 patients in 2006 and 8876 patients in 2008. Average dose was 28 grams every 4 days, with 22 total treatments per patient. Estimated yearly doses used (as 25g/50ml vials) were 318,539 in 2006 and 354,647 in 2008. Manufacturers’ yearly sales were 750,000 and 855,000 vials, respectively. Common reasons for treatment included infection, cancer, and fatigue. Of 9,328 patients for whom data is available, 101 had side effects, mostly minor, including lethargy/fatigue in 59 patients, change in mental status in 21 patients and vein irritation/phlebitis in 6 patients. Publications documented serious adverse events, including 2 deaths in patients known to be at risk for IV vitamin C. Due to confounding causes, the FDA Adverse Events Database was uninformative. Total numbers of patients treated in the US with high dose vitamin C cannot be accurately estimated from this study. Conclusions: High dose IV vitamin C is in unexpectedly wide use by CAM practitioners. Other than the known complications of IV vitamin C in those with renal impairment or glucose 6 phosphate dehydrogenase deficiency, high dose intravenous vitamin C appears to be remarkably safe. Physicians should inquire about IV vitamin C use in patients with cancer, chronic, untreatable, or intractable conditions and be observant of unexpected harm, drug interactions, or benefit.

Background Recombinant interferon treatment can result in several common side effects including fever and injection-site pain. Patients are often advised to use acetaminophen or other over-the-counter pain medications as needed. Little is known regarding the transcriptional changes induced by such co-administration. Methodology/principal findings We tested whether the administration of acetaminophen causes a change in the response normally induced by interferon-beta treatment. CD-1 mice were administered acetaminophen (APAP), interferon-beta (IFN-beta) or a combination of IFN-beta+APAP and liver and serum samples were collected for analysis. Differential gene expression was determined using an Agilent 22 k whole mouse genome microarray. Data were analyzed by several methods including Gene Ontology term clustering and Gene Set Enrichment Analysis. We observed a significant change in the transcription profile of hepatic cells when APAP was co-administered with IFN-beta. These transcriptional changes included a marked up-regulation of genes involved in signal transduction and cell differentiation and down-regulation of genes involved in cellular metabolism, trafficking and the IkappaBK/NF-kappaB cascade. Additionally, we observed a large decrease in the expression of several IFN-induced genes including Ifit-3, Isg-15, Oasl1, Zbp1 and predicted gene EG634650 at both early and late time points. Conclusions/significance A significant change in the transcriptional response was observed following co-administration of IFN-beta+APAP relative to IFN-beta treatment alone. These results suggest that administration of acetaminophen has the potential to modify the efficacy of IFN-beta treatment.

Many heme enzymes show remarkable versatility and atypical kinetics. The fungal extracellular enzyme chloroperoxidase (CPO) characterizes a variety of one and two electron redox reactions in the presence of hydroperoxides. A structural counterpart, found in mammalian microsomal cytochrome P450 (CYP), uses molecular oxygen plus NADPH for the oxidative metabolism (predominantly hydroxylation) of substrate in conjunction with a redox partner enzyme, cytochrome P450 reductase. In this study, we employ the two above-mentioned heme-thiolate proteins to probe the reaction kinetics and mechanism of heme enzymes. Hitherto, a substrate inhibition model based upon non-productive binding of substrate (two-site model) was used to account for the inhibition of reaction at higher substrate concentrations for the CYP reaction systems. Herein, the observation of substrate inhibition is shown for both peroxide and final substrate in CPO catalyzed peroxidations. Further, analogy is drawn in the "steady state kinetics" of CPO and CYP reaction systems. New experimental observations and analyses indicate that a scheme of competing reactions (involving primary product with enzyme or other reaction components/intermediates) is relevant in such complex reaction mixtures. The presence of non-selective reactive intermediate(s) affords alternate reaction routes at various substrate/product concentrations, thereby leading to a lowered detectable concentration of "the product of interest" in the reaction milieu. Occam's razor favors the new hypothesis. With the new hypothesis as foundation, a new biphasic treatment to analyze the kinetics is put forth. We also introduce a key concept of "substrate concentration at maximum observed rate". The new treatment affords a more acceptable fit for observable experimental kinetic data of heme redox enzymes.

BACKGROUND: Safe, cheap and effective adjunct therapies preventing the development of, or reducing the mortality from, severe malaria could have considerable and rapid public health impact. Oral activated charcoal (oAC) is a safe and well tolerated treatment for acute poisoning, more recently shown to have significant immunomodulatory effects in man. In preparation for possible efficacy trials in human malaria, we sought to determine whether oAC would i) reduce mortality due to experimental cerebral malaria (ECM) in mice, ii) modulate immune and inflammatory responses associated with ECM, and iii) affect the pharmacokinetics of parenteral artesunate in human volunteers. \ud \ud METHODS/PRINCIPAL FINDINGS: We found that oAC provided significant protection against P. berghei ANKA-induced ECM, increasing overall survival time compared to untreated mice (p

We found that spadin, a natural peptide derived from sortilin, blocks the mouse TREK-1 channel and might be an efficient and fast-acting antidepressant., Current antidepressant treatments are inadequate for many individuals, and when they are effective, they require several weeks of administration before a therapeutic effect can be observed. Improving the treatment of depression is challenging. Recently, the two-pore domain potassium channel TREK-1 has been identified as a new target in depression, and its antagonists might become effective antidepressants. In mice, deletion of the TREK-1 gene results in a depression-resistant phenotype that mimics antidepressant treatments. Here, we validate in mice the antidepressant effects of spadin, a secreted peptide derived from the propeptide generated by the maturation of the neurotensin receptor 3 (NTSR3/Sortilin) and acting through TREK-1 inhibition. NTSR3/Sortilin interacted with the TREK-1 channel, as shown by immunoprecipitation of TREK-1 and NTSR3/Sortilin from COS-7 cells and cortical neurons co-expressing both proteins. TREK-1 and NTSR3/Sortilin were colocalized in mouse cortical neurons. Spadin bound specifically to TREK-1 with an affinity of 10 nM. Electrophysiological studies showed that spadin efficiently blocked the TREK-1 activity in COS-7 cells, cultured hippocampal pyramidal neurons, and CA3 hippocampal neurons in brain slices. Spadin also induced in vivo an increase of the 5-HT neuron firing rate in the Dorsal Raphe Nucleus. In five behavioral tests predicting an antidepressant response, spadin-treated mice showed a resistance to depression as found in TREK-1 deficient mice. More importantly, an intravenous 4-d treatment with spadin not only induced a strong antidepressant effect but also enhanced hippocampal phosphorylation of CREB protein and neurogenesis, considered to be key markers of antidepressant action after chronic treatment with selective serotonin reuptake inhibitors. This work also shows the development of a reliable method for dosing the propeptide in serum of mice by using AlphaScreen technology. These findings point out spadin as a putative antidepressant of new generation with a rapid onset of action. Spadin can be regarded as the first natural antidepressant peptide identified. It corresponds to a new concept to address the treatment of depression., Author Summary Depression is the most common of psychiatric illnesses, with prevalence estimates ranging from 5% to 20% within the general population. The design of effective treatments for this disorder is a challenging process, and the use of antidepressants has an overall low clinical efficacy as full remission only occurs in one-third of the patients. Moreover, the time between initial treatment and beneficial effects is relatively protracted. These limitations confirm the need to find new biological targets and drugs for the treatment of depression. We recently identified a conserved mouse potassium channel protein called TREK-1 (KCNK2) as a new target for treating depression. Here, we demonstrate that spadin, a natural peptide derived from a propeptide released in blood, is able to block the TREK-1 channel activity and has an antidepressant effect in mouse models of depression. We showed that spadin is an efficient antidepressant in mice that acts much faster (4 d versus several weeks) than fluoxetine, the most commonly used antidepressant. Our results with spadin in mice highlight the potential for novel and more efficacious treatments for depression in humans.

The majority of MDMA (ecstasy) recreational users also consume cannabis. Despite the rewarding effects that both drugs have, they induce several opposite pharmacological responses. MDMA causes hyperthermia, oxidative stress and neuronal damage, especially at warm ambient temperature. However, THC, the main psychoactive compound of cannabis, produces hypothermic, anti-inflammatory and antioxidant effects. Therefore, THC may have a neuroprotective effect against MDMA-induced neurotoxicity. Mice receiving a neurotoxic regimen of MDMA (20 mg/kg x 4) were pretreated with THC (3 mg/kg x 4) at room (21 degrees C) and at warm (26 degrees C) temperature, and body temperature, striatal glial activation and DA terminal loss were assessed. To find out the mechanisms by which THC may prevent MDMA hyperthermia and neurotoxicity, the same procedure was carried out in animals pretreated with the CB(1) receptor antagonist AM251 and the CB(2) receptor antagonist AM630, as well as in CB(1), CB(2) and CB(1)/CB(2) deficient mice. THC prevented MDMA-induced-hyperthermia and glial activation in animals housed at both room and warm temperature. Surprisingly, MDMA-induced DA terminal loss was only observed in animals housed at warm but not at room temperature, and this neurotoxic effect was reversed by THC administration. However, THC did not prevent MDMA-induced hyperthermia, glial activation, and DA terminal loss in animals treated with the CB(1) receptor antagonist AM251, neither in CB(1) and CB(1)/CB(2) knockout mice. On the other hand, THC prevented MDMA-induced hyperthermia and DA terminal loss, but only partially suppressed glial activation in animals treated with the CB(2) cannabinoid antagonist and in CB(2) knockout animals. Our results indicate that THC protects against MDMA neurotoxicity, and suggest that these neuroprotective actions are primarily mediated by the reduction of hyperthermia through the activation of CB(1) receptor, although CB(2) receptors may also contribute to attenuate neuroinflammation in this process.

Background One of the major mechanisms that could produce resistance to antineoplastic drugs in cancer cells is the ATP binding cassette (ABC) transporters. The ABC transporters can significantly decrease the intracellular concentration of antineoplastic drugs by increasing their efflux, thereby lowering the cytotoxic activity of antineoplastic drugs. One of these transporters, the multiple resistant protein 7 (MRP7, ABCC10), has recently been shown to produce resistance to antineoplastic drugs by increasing the efflux of paclitaxel. In this study, we examined the effects of BCR-Abl tyrosine kinase inhibitors imatinib, nilotinib and dasatinib on the activity and expression of MRP7 in HEK293 cells transfected with MRP7, designated HEK-MRP7-2. Methodology and/or Principal Findings We report for the first time that imatinib and nilotinib reversed MRP7-mediated multidrug resistance. Our MTT assay results indicated that MRP7 expression in HEK-MRP7-2 cells was not significantly altered by incubation with 5 µM of imatinib or nilotinib for up to 72 hours. In addition, imatinib and nilotinib (1-5 µM) produced a significant concentration-dependent reversal of MRP7-mediated multidrug resistance by enhancing the sensitivity of HEK-MRP7-2 cells to paclitaxel and vincristine. Imatinib and nilotinib, at 5 µM, significantly increased the accumulation of [3H]-paclitaxel in HEK-MRP7-2 cells. The incubation of the HEK-MRP7-2 cells with imatinib or nilotinib (5 µM) also significantly inhibited the efflux of paclitaxel. Conclusions Imatinib and nilotinib reverse MRP7-mediated paclitaxel resistance, most likely due to their inhibition of the efflux of paclitaxel via MRP7. These findings suggest that imatinib or nilotinib, in combination with other antineoplastic drugs, may be useful in the treatment of certain resistant cancers.

Cyclin A(2) is critical for the initiation of DNA replication, transcription and cell cycle regulation. Cumulative evidences indicate that the deregulation of cyclin A(2) is tightly linked to the chromosomal instability, neoplastic transformation and tumor proliferation. Here we report that treatment of chronic myelogenous leukaemia K562 cells with doxorubicin results in an accumulation of cyclin A(2) and follows by induction of apoptotic cell death. To investigate the potential preclinical relevance, K562 cells were transiently transfected with the siRNA targeting cyclin A(2) by functionalized single wall carbon nanotubes. Knocking down the expression of cyclin A(2) in K562 cells suppressed doxorubicin-induced growth arrest and cell apoptosis. Upon administration with doxorubicin, K562 cells with reduced cyclin A(2) showed a significant decrease in erythroid differentiation, and a small fraction of cells were differentiated along megakaryocytic and monocyte-macrophage pathways. The results demonstrate the pro-apoptotic role of cyclin A(2) and suggest that cyclin A(2) is a key regulator of cell differentiation. To the best of our knowledge, this is the first report that knocking down expression of one gene switches differentiation pathways of human myeloid leukemia K562 cells.

BACKGROUND: Resistance developed by leukemic cells, unsatisfactory efficacy on patients with chronic myeloid leukemia (CML) at accelerated and blastic phases, and potential cardiotoxity, have been limitations for imatinib mesylate (IM) in treating CML. Whether low dose IM in combination with agents of distinct but related mechanisms could be one of the strategies to overcome these concerns warrants careful investigation. METHODS AND FINDINGS: We tested the therapeutic efficacies as well as adverse effects of low dose IM in combination with proteasome inhibitor, Bortezomib (BOR) or proteasome inhibitor I (PSI), in two CML murine models, and investigated possible mechanisms of action on CML cells. Our results demonstrated that low dose IM in combination with BOR exerted satisfactory efficacy in prolongation of life span and inhibition of tumor growth in mice, and did not cause cardiotoxicity or body weight loss. Consistently, BOR and PSI enhanced IM-induced inhibition of long-term clonogenic activity and short-term cell growth of CML stem/progenitor cells, and potentiated IM-caused inhibition of proliferation and induction of apoptosis of BCR-ABL+ cells. IM/BOR and IM/PSI inhibited Bcl-2, increased cytoplasmic cytochrome C, and activated caspases. While exerting suppressive effects on BCR-ABL, E2F1, and beta-catenin, IM/BOR and IM/PSI inhibited proteasomal degradation of protein phosphatase 2A (PP2A), leading to a re-activation of this important negative regulator of BCR-ABL. In addition, both combination therapties inhibited Bruton's tyrosine kinase via suppression of NFkappaB. CONCLUSION: These data suggest that combined use of tyrosine kinase inhibitor and proteasome inhibitor might be helpful for optimizing CML treatment.

Background Postembryonic development, including metamorphosis, of many animals is under control of hormones. In Drosophila and other insects these developmental transitions are regulated by the coordinate action of two principal hormones, the steroid ecdysone and the sesquiterpenoid juvenile hormone (JH). While the mode of ecdysone action is relatively well understood, the molecular mode of JH action remains elusive. Methodology/Principal Findings To gain more insights into the molecular mechanism of JH action, we have tested the biological activity of 86 structurally diverse JH agonists in Drosophila melanogaster. The results were evaluated using 3D QSAR analyses involving CoMFA and CoMSIA procedures. Using this approach we have generated both computer-aided and species-specific pharmacophore fingerprints of JH and its agonists, which revealed that the most active compounds must possess an electronegative atom (oxygen or nitrogen) at both ends of the molecule. When either of these electronegative atoms are replaced by carbon or the distance between them is shorter than 11.5 Å or longer than 13.5 Å, their biological activity is dramatically decreased. The presence of an electron-deficient moiety in the middle of the JH agonist is also essential for high activity. Conclusions/Significance The information from 3D QSAR provides guidelines and mechanistic scope for identification of steric and electrostatic properties as well as donor and acceptor hydrogen-bonding that are important features of the ligand-binding cavity of a JH target protein. In order to refine the pharmacophore analysis and evaluate the outcomes of the CoMFA and CoMSIA study we used pseudoreceptor modeling software PrGen to generate a putative binding site surrogate that is composed of eight amino acid residues corresponding to the defined molecular interactions.

Proteome studies contribute markedly to our understanding of parasite biology, host-parasite interactions, and mechanisms of drug action. For most antimalarial drugs neither mode of action nor mechanisms of resistance development are fully elucidated although this would be important prerequisites for successfully developing urgently required novel antimalarials. Here, we establish a large-scale quantitative proteomic approach to examine protein expression changes in trophozoite stages of the malarial parasite Plasmodium falciparum following chloroquine and artemisinin treatment. For this purpose SIL (stable isotope labeling) using (14)N-isoleucine and (13)C(6),(15)N(1)-isoleucine was optimized to obtain 99% atomic percent enrichment. Proteome fractionation with anion exchange chromatography was used to reduce sample complexity and increase quantitative coverage of protein expression. Tryptic peptides of subfractions were subjected to SCX/RP separation, measured by LC-MS/MS and quantified using the novel software tool Census. In drug treated parasites, we identified a total number of 1,253 proteins, thus increasing the overall number of proteins identified in the trophozoite stage by 30%. A relative quantification was obtained for more than 800 proteins. Under artemisinin and chloroquine treatment 41 and 38 proteins respectively were upregulated (>1.5) whereas 14 and 8 proteins were down-regulated (

BACKGROUND: Obstetric complications have been regarded as a risk factor for schizophrenia later in life. One of the mechanisms underlying the association is postulated to be a hypoxic process in the brain in the offspring around the time of birth. Hippocampus is one of the brain regions implicated in the late-onset dopaminergic dysfunction associated with hypoxic obstetric complications. METHODOLOGY/PRINCIPAL FINDINGS: We used an animal model of perinatal asphyxia, in which rat pups were exposed to 15 min of intrauterine anoxia during Cesarean section birth. At 6 and 12 weeks after birth, the behavior of the pups was assessed using a methamphetamine-induced locomotion test. In addition, the histopathology of the hippocampus was examined by means of stereology. At 6 weeks, there was no change in the methamphetamine-induced locomotion. However, at 12 weeks of age, we found an elevation in methamphetamine-induced locomotor activity, which was associated with an increase of dopamine release in the nucleus accumbens. At the same age, we also found a reduction of the dentate granule cells of the hippocampus. CONCLUSIONS/SIGNIFICANCE: These results suggest that the dopaminergic dysregulation after perinatal asphyxia is associated with a reduction in hippocampal dentate granule cells, and this may partly contribute to the pathogenesis of schizophrenia.

Ginseng, a traditional herbal medicine, may interact with several co-administered drugs in clinical settings, and ginsenosides, the major active components of ginseng, may be responsible for these ginseng-drug interactions (GDIs). Results from previous studies on ginsenosides' effects on human drug-metabolizing P450 enzymes are inconsistent and confusing. Herein, we first evaluated the inhibitory effects of fifteen ginsenosides and sapogenins on human CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 enzymes by using commercially available fluorescent probes. The structure-activity relationship of their effects on the P450s was also explored and a pharmacophore model was established for CYP3A4. Moreover, substrate-dependent phenomena were found in ginsenosides' effects on CYP3A4 when another fluorescent probe was used, and were further confirmed in tests with conventional drug probes and human liver microsomes. These substrate-dependent effects of the ginsenosides may provide an explanation for the inconsistent results obtained in previous GDI reports.