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Phenotypic screening of a 900 compound library of antitubercular nitroimidazole derivatives related to pretomanid against the protozoan parasite Trypanosoma cruzi (the causative agent for Chagas disease) identified several structurally diverse hits with an unknown mode of action. Following initial profiling, a first proof-of-concept in vivo study was undertaken, in which once daily oral dosing of a 7-substituted 2-nitroimidazooxazine analogue suppressed blood parasitemia to low or undetectable levels, although sterile cure was not achieved. Limited hit expansion studies alongside counter-screening of new compounds targeted at visceral leishmaniasis laid the foundation for a more in-depth assessment of the best leads, focusing on both drug-like attributes (solubility, metabolic stability and safety) and maximal killing of the parasite in a shorter timeframe. Comparative appraisal of one preferred lead (58) in a chronic infection mouse model, monitored by highly sensitive bioluminescence imaging, provided the first definitive evidence of (partial) curative efficacy with this promising nitroimidazooxazine class.

Recent advances in the use of transient absorption (TA) and time-resolved vibrational spectroscopies (TRIR and TR3) to study both excited states and reaction intermediates in metal complexes and porphyrins which intercalate into DNA are reviewed. A particularly well-studied class of compounds, which nicely illustrates the comparative advantages of these techniques, is that of ruthenium dppz complexes where the complex might show light-switching or photo-oxidising behaviour depending on the nature of the ancillary ligand. Comparative data on Re- and Cr-dppz complexes are also considered. In the second part of this review transient studies of porphyrins, which are known to intercalate into DNA, are considered with particular emphasis on tetramethyl-pyridiniumporphyrins, where the photophysical behaviour of the metal-free and various metal derivatives are compared.

The performance of photocatalytic treatment processes were assessed using different photocatalysts against E. coli and bacteriophages MS2, ΦX174 and PR772, in a recirculating continuous flow compound parabolic collector system under real sunlight conditions. Suspended TiO2 and ZnO nanoparticle powders and Tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate in solution were tested separately, as well as in combination, using E. coli. For a 3-log reduction of E. coli in distilled water, inactivation rates in terms of cumulative dose were in the order Ru(bpy)3Cl2>(TiO2 & Ru(bpy)3Cl2)>(ZnO & Ru(bpy)3Cl2)>ZnO>TiO2>photolysis. Reactivation of E. coli was observed following all trials despite the detection limit being reached, although the reactivated colonies were observed to be under stress and much slower growing when compared to original colonies. Treatment with Ru(bpy)3Cl2 was also compared against standard photolysis of bacteriophages MS2, ΦX174 and PR772 with the order of photolytic inactivation for a 3-log reduction in terms of cumulative UV-A dose being ΦX174>PR772>MS2. However, MS2 was found to be the most susceptible bacteriophage to treatment with Ru(bpy)3Cl2, with complete removal of the phage observed within the first 15min of exposure. Ru(bpy)3Cl2 also significantly improved inactivation rates for PR772 and ΦX174.

In chronic Trypanosoma cruzi infections, parasite burden is controlled by effective, but nonsterilising immune responses. Infected cells are difficult to detect because they are scarce and focally distributed in multiple sites. However, advances in detection technologies have established a link between parasite persistence and the pathogenesis of Chagas heart disease. Long-term persistence likely involves episodic reinvasion as well as continuous infection, to an extent that varies between tissues. The primary reservoir sites in humans are not definitively known, but analysis of murine models has identified the gastrointestinal tract. Here, we highlight that quantitative, spatial, and temporal aspects of T. cruzi infection are central to a fuller understanding of the association between persistence, pathogenesis, and immunity, and for optimising treatment.

Novel imidazoline benzimidazole derivatives containing diversely substituted phenoxy moieties were synthesized with the aim of evaluating their antitrypanosomal activity, DNA/RNA binding affinity and in vitro ADME properties. The presence of the diethylaminoethyl subunit in 18a–18c led to enhanced antitrypanosomal potency, particularly for 18a and 18c, which contain unsubstituted and methoxy-substituted phenoxy moieties. They were found to be > 2-fold more potent against African trypanosomes than nifurtimox. Fluorescence and CD spectroscopy, thermal denaturation assays and computational analysis indicated a preference of 18a–18c toward AT-rich DNA and their minor groove binding mode. Replacement of the amidine group with less basic and ionisable nitrogen-containing moieties failed to improve membrane permeability of the investigated compounds. Due to structural diversification, the compounds displayed a range of physico-chemical features resulting in variable in vitro ADME properties, leaving space for further optimization of the biological profiles.

The synthesis of several 6,7,8,9,10,11-hexahydro-9-methyl-5,7:9,11-dimethano-5H-benzocyclononen-7-amines is reported. Several of them display low micromolar NMDA receptor antagonist and/or trypanocidal activities. Two compounds are endowed with micromolar anti vesicular stomatitis virus activity, while only one compound shows micromolar anti-influenza activity. The anti-influenza activity of this compound does not seem to be mediated by blocking of the M2 protein. ispartof: Bioorganic & Medicinal Chemistry vol:20 issue:2 pages:942-948 ispartof: location:England status: published

This review considers transient spectroscopic studies of electron transfer reactions between nucleic acids and the excited states of transition metal complexes containing dipyridophenazine or related ligands and focuses mainly on complexes of ruthenium, chromium and rhenium. Particular emphasis is placed on systems where transient UV/visible and/or infrared absorption spectroscopy have been employed.

Graphical abstract, A series of 19 huprines has been evaluated for their activity against cultured bloodstream forms of Trypanosoma brucei and Plasmodium falciparum. Moreover, cytotoxicity against rat myoblast L6 cells was assessed for selected huprines. All the tested huprines are moderately potent and selective trypanocidal agents, exhibiting IC50 values against T. brucei in the submicromolar to low micromolar range and selectivity indices for T. brucei over L6 cells of approximately 15, thus constituting interesting trypanocidal lead compounds. Two of these huprines were also found to be active against a chloroquine-resistant strain of P. falciparum, thus emerging as interesting trypanocidal–antiplasmodial dual acting compounds, but they exhibited little selectivity for P. falciparum over L6 cells.

The synthesis of several (2-oxaadamant-1-yl)amines is reported. They were evaluated as NMDA receptor antagonists and several of them were more active than amantadine, but none was more potent than memantine. None of the tested compounds displayed antiviral activity. Two of the derivatives showed a significant level of trypanocidal activity.

Graphical abstract Several bisnoradamantylamines and noradamantylamines have been synthesized and their antiviral, trypanocidal, NMDA receptor antagonist, and dopamine reuptake inhibitory activities have been studied., The synthesis of several (3-noradamantyl)amines, [(3-noradamantyl)methyl]amines, (3,7-dimethyl-1-bisnoradamantyl)amines, and [(3,7-dimethyl-1-bisnoradamantyl)methyl]amines is reported. They were evaluated against a wide range of viruses and one of them inhibited the cytopathicity of influenza A virus at a concentration similar to that of amantadine. Several of the new polycyclic amines show an interesting activity as NMDA receptor antagonists. A rimantadine analogue displayed significant trypanocidal activity. Moreover, to further characterize the pharmacology of these compounds, their effects on dopamine uptake were also assessed.

Over the last number of years whistleblowers have been gaining prominence. This paper investigates some of the factors that influence the propensity or willingness to blow the whistle among trainee auditors. Three categories of factors are examined: audit firm organisational structures, personal characteristics of whistleblowers and situational variables. A survey of 240 final year students of the Institute of Chartered Accountants in Ireland was undertaken. Trainee auditors (just about to sit their finals) were asked about their confidence in internal and external reporting structures in their firms. Using four scenarios, audit trainees were questioned on their willingness to challenge an audit partner’s inappropriate response to concerns raised during the audit. Finally, audit trainees were asked about the influence of legal protection on their likelihood of whistleblowing. Results indicate that where firms have adequate formal structures for reporting wrongdoing, trainee auditors are more likely to report wrongdoing and have greater confidence that this will not adversely affect their careers. Training increases this confidence. Trainee auditors also express a willingness to challenge an audit partner’s unsatisfactory response to wrongdoing. Significant differences were found in attitudes depending on whether the reports of wrongdoing were internal or external. The willingness to report wrongdoing externally reduces for older (aged over 25) trainees. Not applicable

Z-scan experiments have been performed on solutions of t Bu 4 PcTiO 2 -C 60 ( 2 ) in order to explore a plausible synergistic effect. For comparative purposes we have also measured C 60 and t Bu 4 PcTiO ( 1 ) derivatives. The results indicate poorer excited-state absorption of dyad 2 than those of the corresponding individual phthalocyanine and fullerene components.

The cell surface of the epimastigote form of Trypanosoma cruzi is covered by glycoconjugates rich in galactose. The parasite cannot take up galactose through its hexose transporter, suggesting that the epimerisation of UDP-glucose to UDP-galactose may be the parasite's only route to this sugar. The T. cruzi UDP-glucose 4'-epimerase is encoded by the TcGALE gene. We were unable to make a CL-Brener strain T. cruzi epimastigote TcGALE-/- null mutant, suggesting that the gene is essential. Two TcGALE+/- single-allele knockout clones displayed aberrant morphology and haploid deficiency with respect to galactose metabolism. The morphological phenotypes included shortened flagella, increased incidence of spheromastigotes, agglutination and a novel walnut-like appearance. The reduced supply of UDP-galactose was manifest in the two clones as a six- or nine-fold reduction in the expression of galactopyranose-containing cell surface mucins and negligible or two-fold reduction in the expression of galactofuranose-containing glycoinositolphospholipids. The major loss of mucins as opposed to glycoinositolphospholipids may indicate that the latter are more important for basic parasite survival in culture. The apparent haploid deficiency suggests that epimerase levels are close to limiting, at least in the epimastigote form, and might be exploited as a potential drug target.

Organic reagent assisted self assembly of shaped silver nanoparticles into structured linear arrays and dendritic patterns, is reported. Controlled synthesis of nanoparticle shape is used to prepare color tunable silver nanoparticles. Treatment of the silver nanoparticles with selected organic reagents at certain concentrations enabled the determination of the type of structure or pattern generated as well as control over the length scale over which the assembly is generated. Polyacrylamide treatment of the silver nanoparticles was used to produce linear and branched arrays on the nanometer to micron scale, whereas treatment with thiol compounds enabled the production of dendritic patterns with dimensions ranging from microns to millimeters.

Cells use a variety of signal transduction pathways to coordinate their responses to changes in both the extracellular and intracellular environment. Activation of these pathways is often receptor mediated, resulting in the synthesis of second messenger molecules such as cAMP and cGMP. In Plasmodium falciparum, these cyclic nucleotides have been implicated in regulating sexual differentiation, an obligate stage in the parasite life cycle. cAMP and cGMP are synthesized by adenylyl cyclase and guanylyl cyclase, respectively, and in P. falciparum these enzymes differ significantly from their mammalian counterparts. In this article, we will discuss the properties of the parasite cyclases, with emphasis on their structure, function and evolutionary origin.

We report the characterization of an unusual adenylyl cyclase gene from Plasmodium falciparum, here designated PfACalpha. The level of mRNA expression is maximum during development of gametocytes (the sexual blood stage of the parasite life cycle). The gene is highly interrupted by 22 introns, and reverse transcriptase-PCR analysis revealed that there are multiple mRNA splice variants. One intron has three alternative 3'-splice sites that confer the potential to encode distinct forms of the enzyme using alternative start codons. Deduced amino acid sequences predict membrane-spanning regions, the number of which can vary between two and six depending on the splice variant. Expression of a synthetic form of two of these variants in Xenopus oocytes and in Dictyostelium adenylyl cyclase-deficient mutants, confirms that PfACalpha is a functional adenylyl cyclase. These results identify a novel mechanism in P. falciparum for the generation of multiple isoforms of a key, membrane-bound signaling molecule from a single genomic copy. Comparisons of the catalytic domains of PfACalpha and a second putative P. falciparum adenylyl cyclase (PfACbeta) with those from other species reveal an unexpected similarity with adenylyl cyclases from certain prokaryotes including the cyanobacteria (blue green algae). In addition, the presence of an unusual active site substitution in a position that determines substrate specificity, also characteristic of these prokaryotic forms of the enzyme, further suggests a plastid origin for the Plasmodium cyclases.

It is time to re-check the assumptions of the energy and regulatory economists who propose major changes to traditional regulatory institutions and practices. The time is past due to challenge their vague assumptions about what constitutes effective competition, how to assess the reasonable prospects for such competition in a particular market, and what can and should be done to ensure efficient operations.

Trypanosoma cruzi glutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin. The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract. Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro. The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin. Both tryparedoxin and TcGPXI operate by a ping-pong mechanism. Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides. TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle. Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol. The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites.

NNC-711 [1-(2-((diphenylmethylene)amino)oxy)ethyl)-1,2,4,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride], a γ-aminobutyric acid (GABA) reuptake inhibitor with anticonvulsant activity, was investigated with respect to its cognition-enhancing and neuroprotective potency. In the rat, administration of NNC-711 immediately prior to training prevented amnesia for a passive avoidance task induced by the acetylcholine receptor antagonist scopolamine. NNC-711 was also effective in protecting against ischemia-induced death of CA1 pyramidal neurons in a model of bilateral common carotid artery occlusion in the gerbil. In addition to a neuroprotective activity, NNC-711 exhibited significant cognition-enhancing actions. Daily administration of NNC-711, immediately prior to a spatial learning task, significantly reduced escape latencies in the water maze paradigm in both mature (postnatal day 80) and aged (28 months) rats. All of the above actions exhibited a bell-shaped response with an optimal dose of 0.5–1.0 mg/kg. These investigations with NNC-711 and previous clinical observations on the structurally related anticonvulsant tiagabine confirm the potential of GABA reuptake inhibitors as anti-amnesia and cognition-enhancing agents.

We report here that guanylyl cyclase activity is associated with two large integral membrane proteins (PfGCalpha and PfGCbeta) in the human malaria parasite Plasmodium falciparum. Unusually, the proteins appear to be bifunctional; their amino-terminal regions have strong similarity with P-type ATPases, and the sequence and structure of the carboxyl-terminal regions conform to that of G protein-dependent adenylyl cyclases, with two sets of six transmembrane sequences, each followed by a catalytic domain (C1 and C2). However, amino acids that are enzymatically important and present in the C2 domain of mammalian adenylyl cyclases are located in the C1 domain of the P. falciparum proteins and vice versa. In addition, certain key residues in these domains are more characteristic of guanylyl cyclases. Consistent with this, guanylyl cyclase activity was obtained following expression of the catalytic domains of PfGCbeta in Escherichia coli. In P. falciparum, expression of both genes was detectable in the sexual but not the asexual blood stages of the life cycle, and PfGCalpha was localized to the parasite/parasitophorous vacuole membrane region of gametocytes. The profound structural differences identified between mammalian and parasite guanylyl cyclases suggest that aspects of this signaling pathway may be mechanistically distinct.

The American trypanosome Trypanosoma cruzi is exposed to toxic oxygen metabolites that are generated by drug metabolism and immune responses in addition to those produced by endogenous processes. However, much remains to be resolved about the parasite oxidative defense system, including the mechanism(s) of peroxide reduction. Here we show that reduction of peroxides in T. cruzi is catalyzed by two distinct trypanothione-dependent enzymes. These were localized to the cytosol and mitochondrion. Both are members of the peroxiredoxin family of antioxidant proteins and are characterized by the presence of two conserved domains containing redox active cysteines. The role of these proteins in protecting T. cruzi from peroxide-mediated damage was demonstrated following overexpression of enzyme activity. The parasite-specific features of T. cruzi cytoplasmic peroxiredoxin and T. cruzi mitochondrial peroxiredoxin may be exploitable in terms of drug development.

The parasitic protozoan Trypanosoma cruzi undergoes several differentiation events during its life cycle. Some of these transitions are thought to involve activation of adenylyl cyclase via the binding of peptide ligands to the cell surface. Here we describe the characterisation of the adenylyl cyclase gene family of T. cruzi. Two complete genes and one pseudogene have been sequenced. The protein products appear to have a large extracellular domain, a single transmembrane helix and a cytosolic catalytic domain. The adenylyl cyclase genes are present on at least six chromosomes and are scattered rather than clustered. They form a large polymorphic family in which the extracellular domain is particularly variable. An Escherichia coli adenylyl cyclase mutant could be complemented by expression of the catalytic domain of the T. cruzi enzyme. The recombinant protein had adenylyl cyclase activity in vitro, which was enhanced by increasing concentrations of divalent cations (Mn2+ > Mg2+). This constitutively active recombinant protein will be a useful tool for dissecting the catalytic mechanism of adenylyl cyclase.

Tacrine, one of the drugs available for Alzheimer's disease based on the cholinergic approach, suffers from considerable toxicity. Many analogues of tacrine have been prepared which retain the pharmacologically rich aminopyridine or aminoquinoline motifs. The current research was undertaken to produce an acetylcholinesterase inhibitor by employing 11-aminobenzoquinolizidines (4) and 10-aminobenzoindolizidines (5) as templates. Thus, we aimed to achieve three goals relative to tacrine: eliminate the pyridine and quinoline moieties and render the molecule less flat. Overall, the compounds we prepared were poorer inhibitors of acetylcholinesterase compared to tacrine. The single exception was compound 6f which exhibited an effect comparable to that of tacrine, but only at a dose of the order of 10(-3) M. However, despite the poor acetylcholinesterase inhibition by 6b, this compound proved to be an effective anti-amnesic agent at 45 mg/kg dose.

Rather than viewing it as inevitable that distribution utilities of the future will be large, “wires-only” enterprises, analysts need to spend more time considering the “why” question.

A new type of binuclear Ru(II) complex [Ru(TAP)2(TAP-TAP)Ru(TAP)2]4+ (1) (TAP-TAP=2,2′-bis(1,4,5,8-tetraazaphenanthrene)) produced by photo-reaction of Ru(TAP)32+ in the presence of 5′-guanosine-monophosphate (5′-GMP) has been isolated and characterised. The formation of 1 is proposed to proceed via mono-reduced Ru(TAP)32+, i.e. [Ru2+(TAP)2(TAP⋅−)]+. The emission lifetime of 1 is much greater than that of Ru(TAP)32+, consistent with an increased energy gap between the 3MLCT and 3MC states in 1. This arises because of stabilisation of its 3MLCT state due to the localisation of the excited state electron on the TAP-TAP ligand.

The design of Ru(II) and Os(II) complexes which are photoreactive with deoxyribonucleic acid (DNA) represents one of the main targets for the development of novel molecular tools for the study of DNA and, in the future, for the production of new, metal-based, anti-tumor drugs. In this review, we explain how it is possible to make a complex photoreactive with nucleobases and nucleic acids. According to the photophysical behaviour of the Ru(II) compounds, two types of photochemistry are expected: (1) photosubstitution of a ligand by a nucleobase and another monodentate ligand, which takes place from the triplet, metal-centred (3MC) state; this state is populated thermally from the lowest lying triplet metal to ligand charge transfer (3MLCT) state; (2) photoreaction from the 3MLCT state, corresponding to photoredox processes with DNA bases. The two photoreactivities are in competition. By modulating appropriately the redox properties of the 3MLCT state, an electron transfer process from the base to the excited complex takes place, and is directly correlated with DNA cleavage or the formation of an adduct of the complex to DNA. In this adduct, guanine is linked by N2 to the alpha-position of a non-chelating nitrogen of the polyazaaromatic ligand without destruction of the complex. Different strategies are explained which increase the affinity of the complexes for DNA and direct the complex photoreactivity to sites of special DNA topology or targeted sequences of bases. Moreover, the replacement of the Ru(II) ion by the Os(II) ion in the photoreactive complexes leads to an increased specificity of photoreaction. Indeed, only one type of photoreactivity (from the 3MLCT state) is present for the Os(II) complexes because the 3MC state is too high in energy to be populated at room temperature.

Photoinduced polymerization of cyanoacrylate monomers using a novel class of photoinitiators comprising Group VI metal carbonyl pyridine complexes is reported. Irradiation of a solution containing the monomer and photoinitiator, with visible light (436 nm), leads to a rapid exothermic polymerization reaction which is readily followed by calorimetric techniques. The photoinduced reaction exhibits the features expected of conventional amine initiated polymerization of cyanoacrylates consistent with photo-released pyridine acting as the initiating species.

The genes that encode cruzipain, the major cysteine protease of Trypanosoma cruzi are known to be arranged in tandem arrays. To gain a detailed insight into how these arrays are organised at the chromosomal level we have isolated clones from a cosmid library constructed with DNA from the X10.6 strain. In this strain we found that cruzipain is encoded by two allelic clusters composed of approximately 14 and 23 tandemly repeated genes which are located on homologous chromosomes of 650 and 670 kb. With the exception of the 3'-proximal genes, the cruzipain genes were all of identical or very similar sequence. An unusual feature of the 3'-proximal genes is that they lack the sequences that encode the 130 amino acid carboxyl terminal extension which is characteristic of cruzipain. Both gene clusters are situated in a similar chromosomal environment and are flanked by sequences which have the potential to form Z-DNA. In other eukaryotes, these motifs have been associated with recombinational hotspots and have been demonstrated to enhance gene conversion. The cruzipain genes are transcribed to produce a 1.8-kb transcript which is present at the same steady-state level in each of the parasite life cycle stages. However, protein levels and activity are 4-5-times higher in the insect epimastigote stage than in the trypomastigote and amastigote stages. By implication developmental regulation of cruzipain expression occurs predominantly at the translational and/or post-translational levels.

In the past five years, gene-transfer systems have been established for each of the medically important trypanosomatids: Leishmania sp, Trypanosoma brucei and T. cruzi. Transformation can be mediated by integration, which occurs exclusively by homologous recombination, or by episomal shuttle vectors. In this article, John Kelly will focus on recent progress in the development and applications of trypanosomatid shuttle vectors, ie. vectors which are maintained extrachromosomally and which are capable of autonomous replication in both trypanosomatid and bacterial hosts.

The sub-picosecond deactivation of the thionine singlet state by guanine-containing polynucleotides or mononucleotides has been measured by femtosecond transient absorption spectroscopy. The thionine singlet state was also quenched, although less efficiently, by adenine nucleotides. In contrast, the lifetime of the methylene blue singlet state is only slightly affected by adenine-containing species, although it was rapidly quenched by guanine. A strong correlation was observed between the rates of singlet state deactivation and the Δ G ° values for electron transfer in the dye/nucleotide base systems, although with the thionine/guanine systems no electron transfer products were detected.

Formation of adducts between Ru(TAP)3(2+) (TAP = 1,4,5,8-tetraazaphenanthrene) and DNA has been monitored by gel electrophoresis, UV-vis spectroscopy and dialysis methods. Adduct formation is found for both single- and double-stranded nucleic acids. The reaction with double-stranded DNA is found to be insensitive to solution pH or aeration. Spectroscopic changes similar to those for DNA are found with GMP in oxygen-free pH 5 solution. However, different reactions occur with GMP at higher pH or when the solution contains oxygen. Comparative experiments with double-stranded poly[d(G-C)] or poly[d(A-T)] indicate that the adduct with DNA involves binding to the guanosine moiety. It is proposed that the product is formed by the reaction of the reduced ruthenium complex and oxidised guanine species produced by photo-induced electron transfer.

The light-induced reactions of methylene blue and related phenothiazinium dyes with biological substrates are described. The properties of the excited states of the dyes, their reactions with nucleic acids and their photosensitised chemical modifications of nucleic acid bases are examined. Reports on phenothiazinium dye-induced damage to proteins, lipids, biological membranes, organelles, viruses, bacteria, mammalian cells and carcinomas are reviewed.

Bioluminescence imaging is a non-invasive technique which can be used to monitor infections in real-time. However, its utility is restricted by difficulties in detecting pathogens in deep tissue. 'Red-shifted' luciferases, which emit light of longer wavelength than standard bioluminescence-generating proteins, greatly enhance sensitivity, and have wide applicability for studying parasite infections.

We conducted two experiments to evaluate the flooding dose method for measuring intestinal and liver protein synthesis in sheep. Experiment 1 showed that large doses of phenylalanine did not cause marked metabolic disturbances. Experiment 2 examined the effectiveness of flooding with phenylalanine and the time dependency of the protein synthesis calculation. Rams were injected with 1.2 MBq L-[ring 2,6-3H]phenylalanine/kg body wt and slaughtered 20, 40 or 60 min later. Plasma specific radioactivity reached a plateau within 2.5 min and did not change significantly (P0.05) throughout the experiment. Tissue intracellular free pool specific radioactivity also remained constant from 20 to 60 min postinfusion. Flooding conditions were achieved in the intracellular free pool of intestinal tissues (specific radioactivity 70-96% of plasma specific radioactivity), although liver flooding was less successful (57-67%). Protein synthesis rates measured after 20 min were significantly (P0.05) higher in the liver, jejunum and ileum than those measured at 60 min. Protein synthesis rates also tended to decline with time in the duodenum and colon (P0.05). There were no significant differences (P0.05) between protein synthesis rates calculated using the intracellular specific radioactivity vs. plasma specific radioactivity in the duodenum, ileum or colon. Therefore, this method represents an improvement over continuous infusion methods for measurements of protein synthesis in visceral tissues.

In the genus Leishmania there has been no convincing demonstration of genetic exchange, and it has been proposed that reproduction is clonal. However, preliminary characterization of two strains of Leishmania isolated from wild animals in a zoonotic focus of cutaneous leishmaniasis in the Eastern Province of Saudi Arabia, has suggested that they may represent hybrids of Leishmania major and Leishmania arabica. Evidence presented here strongly supports this hypothesis. Isoenzyme analysis and molecular karyotyping of cloned organisms indicated that the putative hybrids are distinct from other species of Leishmania, and possess characteristics of both L. major and L. arabica. Experiments using highly specific probes demonstrated that kinetoplast minicircle DNA from the putative hybrid contained L. major-specific, but not L. arabica-specific sequences. DNA fingerprinting data obtained using 6 genomic DNA probes were consistent in all cases with a L. major/L. arabica recombinant genotype, and implied both diploidy and allelic segregation. These observations suggest that sexual reproduction may generate genetic diversity within natural Leishmania populations.

Absorption spectra, luminescence spectra and excited-state lifetimes of uranyl ions (UO 2+ 2 ) incorporated in Nafion or Flemion membranes have been studied as a function of hydration. Marked changes in the nature of the spectra and emission lifetime are observed. Thus at 291 K the lifetime changes from 2.0μs in a water-swollen Nafion membrane to 640 μs in a thoroughly dried sample. The lifetime of the dried sample is temperature-invariant, whereas those of heavily hydrated samples show activation energies of 30 ± 2 kJ mol −1 , suggesting that water plays a key role in the excited-state deactivation. A changeover in the decay mechanism is found for samples of intermediate water content. Energy transfer from excited UO 2+ 2 to Eu 3+ is observed, the effect being enhanced by membrane dehydration and being much more pronounced for Flemion membranes than for Nafion. Treatment of the UO 2+ 2 -containing membranes with aqueous KOH causes the formation of products concentrated near the membrane surface. The absorption spectra, luminescence spectra and X-ray diffraction of these materials have been compared with those of the oxides and uranates formed in solutions of various pH. Transmission electron microscopy reveals particles of less than 1 μm in size.

An initiative was launched in 1994 by the Special Programme for Research and Training in Tropical Diseases (TDR) of the WHO to analyse the genomes of the parasites Filaria, Schistosoma, Leishmania, Trypanosoma brucei and Trypanosoma cruzi. Five networks were established through wide publicity, holding meetings of key laboratories and developing proposals which were then reviewed by the Steering Committee of Strategic Research for financial support. The aim of the Programme was to use the platform of these networks to: (1) train scientists from tropical disease-endemic countries; (2) transfer technology and share material and expertise, thereby reducing costs and increasing efficiency; and (3) provide an information system that is accessible globally as soon as the results become available. The initial target was to produce a low-resolution genome map for each of the parasites, but it soon became evident that by using rapidly developing technologies, it might be feasible to complete DNA-sequence analysis for some of the parasites in the next decade, as discussed here by Alberto Carlos Frasch and colleagues, with particular focus on the T. cruzi genome initiative.

The one-day symposium of the London Molecular Parasitology Club (LMPC), held on 8 July 1999, was hosted jointly by the LMPC and the Royal Society of Tropical Medicine and Hygiene. The LMPC, founded ten years ago, organizes monthly meetings at the London School of Hygiene and Tropical medicine on a wide range of topics, spanning a broad spectrum of organisms and experimental approaches. Each meeting aims to discuss at least two parasite systems, with a balance of hard-core molecular science and more biologically focused presentations. If you are interested in speaking at future meetings of the London Molecular Parasitology Club, have suggestions for speakers/topics, or wish to join the e-mailing list, please contact d.smith@ic.ac.uk or john.kelly@Ishtm.ac.uk. These meetings have been sponsored recently by the Wellcome Trust, Pfizer PLC and Hoffman La Roche AG, whose support we gratefully acknowledge.

Fig. 5. Subcellular location of N-GFP in relation to an endoplasmic reticulum-specific marker. Panel 1, a metacyclic trypomastigote stained with TOTO-3 to identify DNA (blue). The intense staining identifies the kinetoplast. Panel 2, the same parasite visualised to identify the endoplasmic reticulum-localised protein BiP. Panel 3, localisation of N-GFP (green). Panel 4, merged images of panels 1–3. Panel 5, phase image. In panel 4, the yellow coloration at the posterior end of the cell indicates where the mitochondrion spirals over and under the endoplasmic reticulum. Bar, 5 mm.

Microparticulate hydrated ruthenium oxide precipitates have been prepared in Nafion ion-exchange membranes by treatment of the Ru-exchanged membranes with KOH or NaOH solutions. These precipitates are concentrated near both membrane surfaces giving a concentration profile which depends on the conditions of preparation. The particle size, nature and distribution across the membrane thickness of the precipitates were characterized by scanning electron microprobe analysis, transmission electron microscopy, electron microdiffraction and X-ray diffraction. The oxide is present as very fine crystalline spherical particles whose size ranges from 200 to 1000 A. Precipitation was also achieved at one side only in free-standing membranes with ruthenium oxide at one side and uranium oxide at the other. Nafion-coated modified electrodes containing precipitated ruthenium oxide particles catalyse the oxidation of water to oxygen and the reduction of oxygen to hydrogen peroxide and water in the presence of mediators. The relevance of these results to the construction of solid polymer electrolyte devices containing active electrocatalysts is discussed.

A decrease of the light transmission with increasing excitation intensity is observed in tetraphenylporphyrin/toluene solutions irradiated with 80 ps pulses at λ=532 nm. The experimental data are compared with simulations using a rate equation model. Excited state absorption cross-sections are evaluated.

The synthesis of chromium and tungsten pentacarbonyl complexes with 4-methyl and 4-phenyl-1,2,4-triazole is reported. The nature of the lowest energy absorption in the ultraviolet spectra of these complexes has been examined and shown to be principally a ligand field band. The triazoles are shown to be acting as monodentate ligands by infrared and 1 H nuclear magnetic resonance spectroscopy. The compounds show strong solvent dependence in the 1 H NMR and these effects are explained by solvent—solute interaction. The temperature dependence of the 1 H NMR spectrum of W(CO) 5 (4-phenyl-1,2,4-triazole) has also been examined.

As the size of the data base nucleic acid sequence information continues to increase, computer methods of sequence data processing will become increasingly important. This paper discusses methods for storage, retrieval, and manipulation of large sequences of bases on a minicomputer using a high-level language, FORTRAN. The merits of treating the genetic code of bases as a simple letters (8 bit bytes) and as integers are considered. Programs have been written and checked that convert or compare nucleic acid and amino acid sequences and animo acid sequences. These programs are available for distribution.

The crystal and molecular structure of tetracarbonyl(6- p -styryl-2,2′-bipyridyl)tungsten(0) have been determined by single crystal X-ray techniques. Cell dimensions are a 13.735(17), b 8.839(6) and c 17.105(12) A for the monoclinic unit cell with β 109.06(8)° and Z = 4. The space group was determined as P 2 1 / n . The structure was solved by conventional Patterson and Fourier methods and refined to a final R -value of 0.055 (1734 unique reflections) by block diagonal least squares. The molecule consists of monomeric units containing one tungsten atom bound to four terminal carbonyl groups and to one 6- p -styryl-2,2′-bipyridyl ligand via its two nitrogen atoms. The presence of the styryl group α to one coordinating nitrogen causes a significant difference in the bond lengths between the central tungsten atom and the nitrogen atoms (WN(1), 2.29(1) A; WN(2) 2.23(1) A).

The photoconductive response of dry, solid calf thymus DNA is measured after excitation with a KrF excimer laser. Both temporal decay of the photoconductive signal and the voltage dependence of the decay show one-dimensional carrier transport characteristics for long strands (5.4 μm long). Short-chain samples (190 nm long) show three-dimensional behaviour with a considerably shorter decay time.

The nonlinear mechanisms for picosecond degenerate four-wave mixing in organic dyes are reviewed and a theoretical description is given. Nonresonant and resonant electronic Kerr effects, saturable absorption and thermally induced refractive index changes are discussed. Experimental studies on a large number of samples are shown and the nonlinear mechanism for each is assessed. In both infrared absorbing dyes (BDN, A9860, S501) and several tetraphenyl porphyrins the dominating mechanism is determined to be thermal in origin. Good agreement between theory and experiment is found in most cases.

M(CO)5(4-VP) (M  Cr, Mo, or W) and M(CO)5 (2-VP) (M  Cr, W), prepared from the reaction of M(CO)5(EtOH) and 2-vinylpyridine (2-VP) or 4-vinyl-pyridine (4-VP), have been characterised by IR, 1H NMR, and UV/visible absorption spectroscope. Copolymers of M(CO)5(VP) with vinylpyridine, styrene, or methyl methacrylate have been synthesised and spectroscopically characterised. Preliminary data on the photosubstitution reactions of W(CO)5−(4-VP) and of its copolymers are presented, and the dependence of the spectroscopic and photochemical properties on the composition of the polymer backbone is reported.

Spectrometric measurements in normal aurora of O2+ first negative (1N) and N2+ first negative (1N) emissions over the wavelength range 5175–5325 A have been obtained with coincident incoherent scatter radar measurements with both instruments pointed in the same direction, the geomagnetic zenith. A comparison of the inferred mean energies derived from the radar observations for several normal aurora was made with the spectral ratio I(O 2 + 1N; 2, 0) I(N 2 + 1N; 0, 3) obtained from the optical observations. The intensity ratio was found to decrease by nearly 40% from aurora with mean energy ∼ 10 keV to aurora with mean energy ∼ 2 keV. The altitude of the peak E-region electron density co-varied with the spectral ratio from ∼ 105 km for the harder aurora to ∼ 130 km for the softer aurora. The inferred rotational temperature from the matching synthetic spectra co-varied from 250 to 500 K over the same energy limits. Model analysis based on both mono-energetic and Maxwellian precipitating electron fluxes show reasonable agreement with the observed I(O 2 + 1N; 2, 0) I(N 2 + 1N; 0, 3) ratio when MSIS n(O2) and n(N2) densities corresponding to the experimental dates are used.

The first stable homoleptic alkenyls of the early transition metals, MRn, (R = C(Ph)=CMe2; M = Ti, Zr, Hf, n = 4; and M = Cr, n = 3) and the related species (C5H5)2MR2 (M = Ti, Zr) and (C5H5)2Zr(Cl)R have been prepared using appropriate organolithium reagents. Cleavage and insertion reactions are reported for the new compounds.