Antiplasmodial activity of the natural product compounds alstonine and himbeline.

Malaria, caused by Plasmodium parasites, continues to be a devastating global health issue. Despite a decline in malaria related deaths over the last decade, overall progress has plateaued. Key challenges to malaria prevention and control include the lack of a broadly effective vaccine and parasite drug resistance, including to the current gold standard artemisinin combination therapies (ACTs). New drugs with unique modes of action are therefore a priority for both the treatment and prevention of malaria. Unlike treatment drugs which need to kill parasites quickly to reduce or prevent clinical symptoms, compounds that kill parasites more slowly may be an option for malaria prevention. Natural products and natural product derived compounds have historically been an excellent source of antimalarial drugs, including the artemisinin component of ACTs. In this study, 424 natural product derived compounds were screened for in vitro activity against P. falciparum in assays designed to detect slow action activity, with 46 hit compounds identified as having >50% inhibition at 10 µM. Dose response assays revealed nine compounds with submicromolar activity, with slow action activity confirmed for two compounds, alstonine and himbeline (50% inhibitory concentration (IC50) 0.17 and 0.58 µM, respectively). Both compounds displayed >140-fold better activity against P. falciparum versus two human cell lines (Selectivity Index (SI) >1,111 and > 144, respectively). Importantly, P. falciparum multi-drug resistant lines showed no cross-resistance to alstonine or himbeline, with some resistant lines being more sensitive to these two compounds compared to the drug sensitive line. In addition, alstonine displayed cross-species activity against the zoonotic species, P. knowelsi (IC50 ~1 µM). Outcomes of this study provide a starting point for further investigations into these compounds as antiplasmodial drug candidates and the investigation of their molecular targets.

Parkinson's disease: Alterations in iron and redox biology as a key to unlock therapeutic strategies.

A plethora of studies indicate that iron metabolism is dysregulated in Parkinson's disease (PD). The literature reveals well-documented alterations consistent with established dogma, but also intriguing paradoxical observations requiring mechanistic dissection. An important fact is the iron loading in dopaminergic neurons of the substantia nigra pars compacta (SNpc), which are the cells primarily affected in PD. Assessment of these changes reveal increased expression of proteins critical for iron uptake, namely transferrin receptor 1 and the divalent metal transporter 1 (DMT1), and decreased expression of the iron exporter, ferroportin-1 (FPN1). Consistent with this is the activation of iron regulator protein (IRP) RNA-binding activity, which is an important regulator of iron homeostasis, with its activation indicating cytosolic iron deficiency. In fact, IRPs bind to iron-responsive elements (IREs) in the 3ꞌ untranslated region (UTR) of certain mRNAs to stabilize their half-life, while binding to the 5ꞌ UTR prevents translation. Iron loading of dopaminergic neurons in PD may occur through these mechanisms, leading to increased neuronal iron and iron-mediated reactive oxygen species (ROS) generation. The "gold standard" histological marker of PD, Lewy bodies, are mainly composed of α-synuclein, the expression of which is markedly increased in PD. Of note, an atypical IRE exists in the α-synuclein 5ꞌ UTR that may explain its up-regulation by increased iron. This dysregulation could be impacted by the unique autonomous pacemaking of dopaminergic neurons of the SNpc that engages L-type Ca+2 channels, which imparts a bioenergetic energy deficit and mitochondrial redox stress. This dysfunction could then drive alterations in iron trafficking that attempt to rescue energy deficits such as the increased iron uptake to provide iron for key electron transport proteins. Considering the increased iron-loading in PD brains, therapies utilizing limited iron chelation have shown success. Greater therapeutic advancements should be possible once the exact molecular pathways of iron processing are dissected.

Maculotoxin: a neurotoxin from the venom glands of the octopus Hapalochlaena maculosa identified as tetrodotoxin.

Maculotoxin, a potent neurotoxin isolated from the posterior salivary glands of the blue-ringed octopus. Hapalochlaena maculosa, has now been identified as tetrodotoxin. This is the first reported case in which tetrodotoxin has been found to occur in a venom.

Fluorine Is a Major Constituent of the Marine Sponge Halichondria moorei.

Fluorine constitutes about 10 percent of the dry weight of the marine sponge Halichondria moorei. The fluorine occurs as potassium fluorosilicate, which is a potent anti-inflammatory agent. A closely related sponge living in the same habitat does not contain any fluorine. The habitat was found to be free of fluorine except for the small amount naturally present in seawater.

Evaluation of fermentation conditions triggering increased antibacterial activity from a near-shore marine intertidal environment-associated Streptomyces species.

A near-shore marine intertidal environment-associated Streptomyces isolate (USC-633) from the Sunshine Coast Region of Queensland, Australia, cultivated under a range of chemically defined and complex media to determine optimal parameters resulting in the secretion of diverse array of secondary metabolites with antimicrobial properties against various antibiotic resistant bacteria. Following extraction, fractioning and re-testing of active metabolites resulted in persistent antibacterial activity against Escherichia coli (Migula) (ATCC 13706) and subsequent Nuclear Magnetic Resonance (NMR) analysis of the active fractions confirmed the induction of metabolites different than the ones in fractions which did not display activity against the same bacterial species. Overall findings again confirmed the value of One Strain-Many Compounds (OSMAC) approach that tests a wide range of growth parameters to trigger bioactive compound secretion increasing the likelihood of finding novel therapeutic agents. The isolate was found to be adaptable to both marine and terrestrial conditions corresponding to its original near-shore marine intertidal environment. Wide variations in its morphology, sporulation and diffusible pigment production were observed when different growth media were used.

Stimulating the proliferation, migration and lamellipodia of Schwann cells using low-dose curcumin.

Transplantation of peripheral glia is being trialled for neural repair therapies, and identification of compounds that enhance the activity of glia is therefore of therapeutic interest. We have previously shown that curcumin potently stimulates the activity of olfactory glia. We have now examined the effect of curcumin on Schwann cell (SC) activities including proliferation, migration and the expression of protein markers. SCs were treated with control media and with different concentrations of curcumin (0.02-20 µM). Cell proliferation was determined by MTS assay and migration changes were determined by single live cell migration tracking. We found that small doses of curcumin (40 nM) dramatically increased the proliferation and migration in SCs within just one day. When compared with olfactory glia, curcumin stimulated SC proliferation more rapidly and at lower concentrations. Curcumin significantly increased the migration of SCs, and also increased the dynamic activity of lamellipodial waves which are essential for SC migration. Expression of the activated form of the MAP kinase p38 (p-p38) was significantly decreased in curcumin-treated SCs. These results show that curcumin's effects on SCs differ remarkably to its effects on olfactory glia, suggesting that subtypes of closely related glia can be differentially stimulated by curcumin. Overall these results demonstrate that the therapeutically beneficial activities of glia can be differentially enhanced by curcumin which could be used to improve outcomes of neural repair therapies.

Synthesis and structure-activity relationship of pyrazolo[3,4-d]pyrimidines: potent and selective adenosine A1 receptor antagonists.

A series of 12 substituted 1-phenylpyrazolo[3,4-d]pyrimidines were synthesized and evaluated for rat brain adenosine A1 and A2a receptor binding affinity. Substituents at C-4 and C-6 were varied in order to define these regions in terms of molecular recognition by the receptor subtypes. At C-4, the effects of a mercapto, methylthio, and amino substituent were evaluated, while at C-6, amides with varying alkyl groups extending from the alpha-carbon were examined. This study identified both potent and selective adenosine A1 receptor antagonists. The most potent of the 12 compounds was alpha-[(4-amino-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl)thio]hexanamide (14); with an A1 Ki of 0.939 nM and an A2a Ki of 88.3 nM, this compound is 94-fold A1 selective. The most selective of the 12 compounds was alpha-[[4-(methylthio)-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl]thio]hex anamide (10); with an A1 Ki of 6.81 nM and an A2a Ki > 40 000 nM, this compound is > 5900-fold A1 selective. The structure-activity relationships for the complete series has identified discrete structural differences between the A1 and A2a receptors with respect to the binding of pyrazolo[3,4-d]pyrimidines. This study resulted in prediction that increased A1 affinity could be achieved by incorporation of NH-alkyl substituents at C-4. This was confirmed by synthesis of alpha-[[4-(methylamino)-1-phenylpyrazolo[3,4-d]pyrimidin-6-yl]thiol] hexanamide (15) which was found to have an A1 Ki of 0.745 nM.

The three binding domain model of adenosine receptors: molecular modeling aspects.

Using molecular modeling, adenosine receptor ligands were fitted together to maximize correlations between the three most important factors controlling binding to the receptor, namely steric, hydrophobic, and electrostatic complimentarily. Structure-activity relationships can be explained by three binding domains on the receptors. These are hydrophobic, aromatic, and ribose binding domains. We propose that the N6, C2, and C8 hydrophobic binding domains are not discreet but occupy the same region of the receptor.

Synthesis and adenosine receptor affinity of a series of pyrazolo[3,4-d]pyrimidine analogues of 1-methylisoguanosine.

Pyrazolo[3,4-d]pyrimidines are pyrazolo analogues of purines. They have been shown to be a general class of compounds which exhibit A1 adenosine receptor affinity. Two series of pyrazolo[3,4-d]pyrimidine analogues of 1-methylisoguanosine have been synthesized. The first involved substitution of the N1-position while the second involved substitution of the N5-position. Both alkyl and aryl substituents were examined. All compounds were tested for A1 adenosine receptor affinity by using a (R)-[3H]-N6-(phenylisopropyl)adenosine binding assay. The 3-chlorophenyl group showed the greatest activity in the N1-position and the butyl group produced the greatest activity in the N5-position. Combination of the best substituent in each of these positions enhanced the overall activity. The most potent compound was 4-amino-5-N-butyl-1-(3-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-6(5H)- one with an IC50 of 6.4 x 10(-6) M. Selectivity at the receptor subclasses was examined by performing an A2 adenosine receptor affinity assay with [3H]CGS 21680. This series of compounds were slightly less potent at A2 receptors. 4-Amino-5-N-butyl-1-(3-chlorophenyl-1H-pyrazolo[3,4-d]pyrimidin-6(5H)-one was the most potent compound with an IC50 of 19.2 x 10(-6) M.

Poor technetium-99m-DMSA renal uptake with near normal technetium-99m-DTPA uptake caused by tubulointerstitial renal disease.

We present a patient with tubulointerstitial renal disease and poor renal 99mTc-DMSA uptake. A 99mTc-DTPA scan was normal and the creatinine clearance only minimally decreased. In this case, 99mTc-DMSA uptake did not correlate with "global renal function," but rather with the functioning tubular mass.

A decision analysis approach to the treatment of patients with suspected pulmonary emboli and an intermediate probability lung scan.

There remains no clear consensus as to the appropriate further investigation and management of the patient suspected of pulmonary embolism (PE) who has an intermediate lung scan. Clinical assessment is documented as unreliable, yet many of these patients are unlikely to be treated or to have further tests despite a 36% chance of having PE. Using Medical Decision Analysis, four management strategies for such patients have been examined in terms of mortality and morbidity up to 6 mo post-presentation. The strategies were: (1) treat all patients; (2) treat no patients; (3) perform pulmonary angiography; and (4) perform contrast venography. In the last two strategies, the patients with positive examinations are treated; those with negative examinations are not treated. An extensive literature review was performed to provide probability estimates of chance events and outcomes. If all patients are treated, there is 96.8% chance of survival, with an 85.8% chance of survival with no major complications. If no patients are treated, survival is 89.3% and complication-free survival is 89.3%. Angiography and venography results were 96.7%, 93.1% and 94.6% and 89.6%, respectively. We conclude that in patients suspected of PE who have intermediate lung scan results, the optimal strategy is pulmonary angiography since this results in the highest survival with the lowest complications.

Incidence of pulmonary embolism in single segmental mismatch on lung scanning.

UNLABELLED: Controversy exists as to whether patients with single segmental mismatch (SSM) on a ventilation/perfusion (VQ) lung scan should be given a low or an intermediate probability of pulmonary embolism (PE). METHODS: Pulmonary angiography was used to evaluate the incidence of PE in SSM at the authors' institution. From January 1991 to January 1993, 1449 VQ scans were performed. RESULTS: With modified Biello criteria, 283 were high probability; 628, low probability; 273, normal; and 273, intermediate probability. Of the intermediate probability scans, 61 had SSM. Forty of these patients underwent pulmonary angiography. Twelve patients had PE in the area of the SSM, giving an incidence of PE of 30%. The risk of PE in SSM in the different lung regions was also analyzed. Twenty-three SSM were in the bases of the lung with a 22% incidence of PE; 17 SSM were either in the midzone or apex with a 41% incidence of PE (p = not significant). CONCLUSION: SSM carries a 30% risk of PE. Accordingly, SSM should be given an intermediate probability of PE and not a low probability of PE.

Detection of postoperative deep-venous thrombosis using technetium-99m-labeled tissue plasminogen activator.

UNLABELLED: The current noninvasive methods of deep-venous thrombosis (DVT) detection in the asymptomatic patient are sufficiently inaccurate so as to preclude their routine use. This present study reports the accuracy of scintigraphic scanning with 99mTc-rt-PA in asymptomatic postoperative patients using contrast venography as the gold standard. METHODS: Fifty-three consecutive postarthroplasty patients (30 THR, 23 TKR) (16 women, 37 men; mean age 71 yr; range 52-85 yr) underwent scintigraphic scanning with 99mTc-rt-PA and contrast venography, on the operated leg, in order to assess the accuracy of this new technique in these asymptomatic patients. RESULTS: Eighty-four segments were of diagnostic quality on contrast venography. Of the 15 thrombosed segments, 14 had positive scans. In the 69 nonthrombosed segments, 63 had negative scans. Thus, scintigraphic scanning with 99mTc-rt-PA had a sensitivity of 93% and a specificity of 91%. CONCLUSION: This study demonstrated that scintigraphic scanning with modified 99mTc-rt-PA is accurate in the detection of DVT in patients undergoing total hip or total knee arthroplasty.

Technetium-99m-modified recombinant tissue plasminogen activator to detect deep venous thrombosis.

UNLABELLED: We report a method for deep venous thrombosis (DVT) detection which uses 99mTc-labeled modified recombinant tissue plasminogen activator (rt-PA) scintigraphy. A Phase III clinical trial was performed on 79 patients with suspected DVT. METHODS: The plasminogen binding site of rt-PA was permanently inhibited without inactivating the fibrin binding site. The modified molecule was radiolabeled with 99mTc. Scintigraphy was performed and the results were compared to those of contrast venography. RESULTS: Of 14 thrombosed proximal segments, 13 had positive scans; in the 53 nonthrombosed proximal segments, 49 had negative scans. In proximal vein thrombosis, rt-PA scintigraphy had a sensitivity of 93% and a specificity of 92%. Of the 36 thrombosed calf vein segments, 31 had positive scans; in the 30 nonthrombosed calf segments, 28 had negative scans. In calf vein thrombosis, scanning has a sensitivity of 86% and a specificity of 93%. CONCLUSIONS: Scintigraphic scanning with this 99mTc modified rt-PA permits accurate detection of thrombus in both proximal and calf veins in patients with clinically suspected DVT. The technique detects both fresh and aged thrombi and is unaffected by heparin administration. Further study in other patient groups is needed to define the overall clinical utility.

Meningitis caused by Streptococcus dysgalactiae in a preterm infant.

This report describes a case of meningitis caused by a Lancefield group C streptococcus (Streptococcus dysgalactiae) in a 9-week-old infant. Bacteria of this group rarely cause serious infections in man. The organism was identified as a member of Lancefield group C by the acid extraction method and as S. dysgalactiae by biochemical tests. The patient's condition responded well to penicillin and tobramycin therapy, with no obvious neurologic sequelae.

Preferences for health outcomes. Comparison of assessment methods.

This study compared standard gamble (SG), time trade-off (TTO), and category scaling (CS) methods for assessing preferences among hypothetical outcomes of coronary artery bypass surgery. High correlations among assessment methods, as found in some previous studies, do not assure the absence of systematic differences in rating obtained by different methods. This study used analysis of variance to test for differences among the three assessment methods. Questionnaire responses were obtained from 67 of 109 physicians participating in a postgraduate course on clinical decision making, following a lecture and workshop on utility theory. SG and CS were used to rate multivariate combinations of angina (none, moderate, and severe) and survival (0, 5, and 10 years); and SG, TTO, and CS were used to rate univariate outcomes with angina (none, moderate, and severe) for the remainder of their life expectancy. SG ratings were higher than TTO ratings, which were higher than CS ratings (p less than 0.001 for all comparisons). Multivariate responses revealed a significant interaction between angina and survival dimensions using CS, but not using SG. We conclude that these methods are not interchangeable and that differences between SG and CS require a more complex explanation than differences in attitude toward risk.

Anhydride modified cantharidin analogues. Is ring opening important in the inhibition of protein phosphatase 2A?

A series of anhydride modified cantharidin analogues have been synthesised and screened for their ability to inhibit protein phosphatase 2A. Surprisingly only analogues capable of undergoing a facile ring opening of the anhydride moiety displayed any significant inhibition. Subsequent NMR experiments indicated that 7-oxobicyclo[2.2.1]heptane-2,3-dicarboxylic acid was the major (sole) species under assay conditions. The ability of these modified anhydro-cantharidin analogues to inhibit protein phosphatase 2A varies from 4 (16) to 100% (8) at 100 microM test concentration.

A study of the binding requirements of calyculin A and dephosphonocalyculin A with PP1, development of a molecular recognition model for the binding interactions of the okadaic acid class of compounds with PP1.

The interactions of the okadaic acid class of compounds, with special emphasis on the solution structures of calyculin A and dephosphonocalyculin A with PP1 are reported. After examination of the interactions of all docked structures, a receptor based pharmacophore model for the interactions of the protein phosphatase inhibitors has been developed. Calyculin A or dephosphonocalyculin A can interact with the enzyme in either a manner similar to the reported crystal structure, or in an extended form. The inhibitors require two essential regions interacting with the hydrophobic region and the central metal binding regions of the enzyme. This simplified model is consistent with previously published models of the okadaic acid class of compounds with PP1.

Attitudes of general practitioners to their interactions with pharmaceutical companies: a qualitative study.

OBJECTIVES: To obtain the attitudes of a sample of General Practitioners to their interactions with pharmaceutical companies. DESIGN: Semi-structured face to face interviews. SETTING: General Practices in the north-west of Ireland. RESULTS: General Practitioners do not value their interactions with pharmaceutical representatives. They regard it as promotional, not educational activity and believe they are presented with biased information. Positive aspects (social, and receiving information) do not compensate. The content of educational meetings should be decided by G.Ps. alone. More directly promotional meetings are valued less, except when fairly lavish, in which case they are harder to resist. Material received through the post is not valued at all by G.Ps. CONCLUSIONS: Pharmaceutical companies in Ireland have a good relationship with G.Ps. It is in jeopardy. To rescue it, companies need to provide G.Ps. with assistance (information and other types) which is directly helpful to G.Ps. caring for their patients. Companies need to row back on the deluge of promotional material that G.Ps. are faced with. G.Ps. need to be trained to learn how to demand more helpful material from companies, and to refuse the promotional tidal wave.

Needle biopsy of the pleura in the diagnosis of pleural effusion.

Needle biopsy of the parietal pleura was undertaken in 64 patients with undiagnosed pleural effusion. An adequate specimen was obtained in 96% of procedures. This was diagnostic in 45% of those due to malignancy and in 50% of those due to tuberculosis. A second biopsy improved the combined diagnostic yield in these two diseases from 32% to 46%. Pleural fluid cytology was unhelpful in establishing the presence of a malignancy, and culture of the biopsy specimen was helpful in one case.