Infective stages of Leishmania in the sandfly vector and some observations on the mechanism of transmission

Infective stages of Leishmania (Leishmania) amazonensis, capable of producing amastigote infections in hamster skin, were shown to be present in the experimentally infected sandfly vector Lutzomyia flaviscutellata 15, 25, 40, 49, 70, 96 and 120 hours after the flies had received their infective blood-meal. Similarly, infective stages of Leishmania (L.) chagasi were demonstrated in the experimentally infected vector Lu. longipalpis examined 38, 50, 63, 87, 110, 135, 171 and 221 hours following the infective blood-meal, by the intraperitoneal inoculation of the flagellates into hamsters. The question of whether or not transmission by the bite of the sandfly is dependent on the presence of [quot ]metacyclic[quot ] promastigotes in the mouthparts of the vector is discussed.

Anti-schistosomal drugs: observations on the mechanism of drug resistance to hycanthone, and on the involvement of host antibodies in the mode of action of praziquantel

This paper reports recent observations from our laboratory dealing with the anti-schistosome drugs hycanthone (HC) and praziquantel (PZQ). In particular, we discuss a laboratory model of drug resistance to HC in Schistosoma mansoni and show that drug sensitive and resistant lines of the parasite can be differentiated on the basis of restriction fragment length polymorphisms using homologous ribosomal gene probes. In addition, we summarize data demonstrating that effective chemotherapy of S. mansoni infection with PZQ in mice requires the presence of host anti-parasite antibodies. These antibodies bind to PZQ treated worms and may be involved in an antibody-dependent cellular cytotoxicity reactions which result in the clearance of worms from the vasculature.

Mechanism of action of a nitroimidazole-thiadiazole derivate upon Trypanosoma cruzi tissue culture amastigotes

Megazol (CL 64,855) a very effective drug in experimental infections by Trypanosoma cruzi, and also in in vitro assays with vertebrate forms of the parasite, had its parasite, had its activity upon macromolecule biosynthesis tested using tissue culture-derived amastigote forms. Megazol presented a drastic inhibition of [3H]-uridine incorporation, suggesting a selective activity upon protein synthesis. Comparing the three drugs, megazol was more potent than nifurtimox and benznidazole in inhibiting protein an DNA synthesis. Megazol showed a 91% of inhibition of [3H]-leucine incorporation whereas nifurtimox and benznidazole, 0% and 2%, respectively. These latter two drugs inhibited the incorporation of all the precursors tested at similar levels, but the concentration of benznidazole was always three times higher, suggesting different mechanisms of action or, more probably, a greater efficiency of the 5-nitrofuran derivate in relation to the 2-nitroimidazole. So, wes conclude that the mode of action of megazol is different from the ones of nifurtimox and benznidazole and that its primary effect is associated with an impairment of protein synthesis.

RecA-mediated annealing of single-stranded DNA and its relation to the mechanism of homologous recombination.

We demonstrate that RecA protein can mediate annealing of complementary DNA strands in vitro by at least two different mechanisms. The first annealing mechanism predominates under conditions where RecA protein causes coaggregation of single-stranded DNA (ssDNA) molecules and where RecA-free ssDNA stretches are present on both reaction partners. Under these conditions annealing can take place between locally concentrated protein-free complementary sequences. Other DNA aggregating agents like histone H1 or ethanol stimulate annealing by the same mechanism. The second mechanism of RecA-mediated annealing of complementary DNA strands is best manifested when preformed saturated RecA-ssDNA complexes interact with protein-free ssDNA. In this case, annealing can occur between the ssDNA strand resident in the complex and the ssDNA strand that interacts with the preformed RecA-ssDNA complex. Here, the action of RecA protein reflects its specific recombination promoting mechanism. This mechanism enables DNA molecules resident in the presynaptic RecA-DNA complexes to be exposed for hydrogen bond formation with DNA molecules contacting the presynaptic RecA-DNA filament.

On the mechanism of protective action of cold acclimatization against carbon tetrachloride - and ethionine-induced fatty liver

In this study the hepatic lipoprotein lipase (LPL), activity was evaluated in adult female mice acclimatized at 5-C and submitted to carbon tetrachloride (CCI) or ethionine, in order to determine the possible role of this enzuyme in the fatty liver. The results were compared with those obtained in mice kept at room temperature (27-C) that the same hepatoesteatosis inducing agent. In contrast to animals kept at room temperature, in cold aclimatized mice neither the enhancement of the LPL-liver activity by the action of CCI or ethionine occurred nor the development of fatty infiltration in the liver was observed. We conclude that the low temperature induced a protective effect against CCI or ethionine-induced fatty liver that was correlated with the no-increase of the hepatic LPL activity.

Mechanism of action of Bacillus thuringiensis toxins

The selectivity of Bacillus thuringiensis toxins is determined both by the toxin structure and by factors inherent to the insect. These toxins contain distinct domains that appear to be functionally important in toxin binding to protein receptors in the midgut of susceptible insects, and the subsequent formation of a pore in the insect midgut epithelium. In this article features necessary for the insecticidal activity of these toxins are discussed. These include toxin structure, toxin processing in the insect midgut, the identification of toxin receptors in susceptible insects, and toxin pore formation in midgut cells. In addition a number of B. thuringiensis toxins act synergistically to exert their full insecticidal activity. This synergistic action is critical not only for expressing the insecticidal activity of these toxins, but could also play a role in delaying the onset of insect resistance.

A geometric mechanism of diffusion: Rigorous verification in a priori unstable Hamiltonian systems

In this paper we consider a representative a priori unstable Hamiltonian system with 2+1/2 degrees of freedom, to which we apply the geometric mechanism for diffusion introduced in the paper Delshams et al., Mem.Amer.Math. Soc. 2006, and generalized in Delshams and Huguet, Nonlinearity 2009, and provide explicit, concrete and easily verifiable conditions for the existence of diffusing orbits. The simplification of the hypotheses allows us to perform explicitly the computations along the proof, which contribute to present in an easily understandable way the geometric mechanism of diffusion. In particular, we fully describe the construction of the scattering map and the combination of two types of dynamics on a normally hyperbolic invariant manifold.

Immunological mechanism of action and clinical profile of disease-modifying treatments in multiple sclerosis.

Multiple sclerosis (MS) is a life-long, potentially debilitating disease of the central nervous system (CNS). MS is considered to be an immune-mediated disease, and the presence of autoreactive peripheral lymphocytes in CNS compartments is believed to be critical in the process of demyelination and tissue damage in MS. Although MS is not currently a curable disease, several disease-modifying therapies (DMTs) are now available, or are in development. These DMTs are all thought to primarily suppress autoimmune activity within the CNS. Each therapy has its own mechanism of action (MoA) and, as a consequence, each has a different efficacy and safety profile. Neurologists can now select therapies on a more individual, patient-tailored basis, with the aim of maximizing potential for long-term efficacy without interruptions in treatment. The MoA and clinical profile of MS therapies are important considerations when making that choice or when switching therapies due to suboptimal disease response. This article therefore reviews the known and putative immunological MoAs alongside a summary of the clinical profile of therapies approved for relapsing forms of MS, and those in late-stage development, based on published data from pivotal randomized, controlled trials.

Electron cryo-microscopy reveals mechanism of action of propranolol on artificial membranes.

The pharmacological activity of several amphiphilic drugs is often related to their ability to interact with biological membranes. Propranolol is an efficient multidrug resistance (MDR) modulator; it is a nonselective beta-blocker and is thought to reduce hypertension by decreasing the cardiac frequency and thus blood pressure. It is used in drug delivery studies in order to treat systemic hypertension. We are interested in the interaction of propranolol with artificial membranes, as liposomes of controllable size are used as biocompatible and protective structures to encapsulate labile molecules, such as proteins, nucleic acids or drugs, for pharmaceutical, cosmetic or chemical applications. We present here a study of the interaction of propranolol, a cationic surfactant, with pure egg phosphatidylcholine (EPC) vesicles. The gradual transition from liposome to micelle of EPC vesicles in the presence of propranolol was monitored by time-resolved electron cryo-microscopy (cryo-EM) under different experimental conditions. The liposome-drug interaction was studied with varying drug/lipid (D/L) ratios and different stages were captured by direct thin-film vitrification. The time-series cryo-EM data clearly illustrate the mechanism of action of propranolol on the liposome structure: the drug disrupts the lipid bilayer by perturbing the local organization of the phospholipids. This is followed by the formation of thread-like micelles, also called worm-like micelles (WLM), and ends with the formation of spherical (globular) micelles. The overall reaction is slow, with the process taking almost two hours to be completed. The effect of a monovalent salt was also investigated by repeating the lipid-surfactant interaction experiments in the presence of KCl as an additive to the lipid/drug suspension. When KCl was added in the presence of propranolol the overall reaction was the same but with slower kinetics, suggesting that this monovalent salt affects the general lipid-to-micelle transition by stabilizing the membrane, presumably by binding to the carbonyl chains of the phosphatidylcholine.

Unique genome replication mechanism of the archaeal virus AFV1.

The exceptional genomic content and genome organization of the Acidianus filamentous virus 1 (AFV1) that infects the hyperthermophilic archaeon Acidianus hospitalis suggest that this virus might exploit an unusual mechanism of genome replication. An analysis of replicative intermediates of the viral genome by two-dimensional (2D) agarose gel electrophoresis revealed that viral genome replication starts by the formation of a D-loop and proceeds via strand displacement replication. Characterization of replicative intermediates using dark-field electron microscopy, in combination with the 2D agarose gel electrophoresis data, suggests that recombination plays a key role in the termination of AFV1 genome replication through the formation of terminal loops. A terminal protein was found to be attached to the ends of the viral genome. The results allow us to postulate a model of genome replication that relies on recombination events for initiation and termination.

High-dose intravenous immunoglobulin treatment and cerebral vasospasm : a possible mechanism of ischemic encephalopathy?

A 46-year-old woman with a severe polyradiculoneuropathy treated with high-dose intravenous immunoglobulin (IVIg) presented an encephalopathy with increased blood flow velocities of the middle cerebral arteries (MCAs) detected by transcranial Doppler (TCD) studies. The similitude between this observation and another case recently reported of a patient suffering from Guillain-Barré syndrome (GBS) and cerebral blood flow abnormalities after IVIg treatment prompted us to investigate the responsibility of the IVIg therapy in the genesis of these blood flow alterations. We studied therefore by TCD 10 consecutive patients who underwent this treatment for different reasons. In 1 case we observed an asymptomatic, spontaneously reversible increase in the blood flow velocities of the MCAs consistent with a vasospasm and occurring 3-10 days after completion of the therapy. Stroke and ischemic encephalopathy have been reported as possible complications of IVIg treatment. In the case under discussion, clinical events appeared shortly after the administration of the IVIg therapy and responded favorably to a treatment with nimodipine. Other etiopathogenic mechanisms, in particular a CNS vasculopathic process related to the GBS itself, have to be considered as well. Further studies, with a larger number of patients, are therefore needed to evaluate the underlying mechanisms of blood flow abnormalities occurring sometimes in GBS patients after IVIg treatment.

Molecular mechanism of myosin Va recruitment to dense core secretory granules.

The brain-spliced isoform of Myosin Va (BR-MyoVa) plays an important role in the transport of dense core secretory granules (SGs) to the plasma membrane in hormone and neuropeptide-producing cells. The molecular composition of the protein complex that recruits BR-MyoVa to SGs and regulates its function has not been identified to date. We have identified interaction between SG-associated proteins granuphilin-a/b (Gran-a/b), BR-MyoVa and Rab27a, a member of the Rab family of GTPases. Gran-a/b-BR-MyoVa interaction is direct, involves regions downstream of the Rab27-binding domain, and the C-terminal part of Gran-a determines exon specificity. MyoVa and Gran-a/b are partially colocalised on SGs and disruption of Gran-a/b-BR-MyoVa binding results in a perinuclear accumulation of SGs which augments nutrient-stimulated hormone secretion in pancreatic beta-cells. These results indicate the existence of at least another binding partner of BR-MyoVa that was identified as rabphilin-3A (Rph-3A). BR-MyoVa-Rph-3A interaction is also direct and enhanced when secretion is activated. The BR-MyoVa-Rph-3A and BR-MyoVa-Gran-a/b complexes are linked to a different subset of SGs, and simultaneous inhibition of these complexes nearly completely blocks stimulated hormone release. This study demonstrates that multiple binding partners of BR-MyoVa regulate SG transport, and this molecular mechanism is universally used by neuronal, endocrine and neuroendocrine cells.

Radio Frequency Identification Tagging as a Mechanism of Creating a Viable Producer’s Brand in the Cattle Industry, May 2005

This manuscript reports on a project to examine the feasibility of extensive radio frequency identification (RFID) tagging to determine product provenance in the meat production industry. The investigators examined existing technologies and meat production processes as well as emerging technologies in RFID tagging to assess the potential of RFID technologies for provenance assurance. While RFID technologies hold tremendous promise for traceability, the current state of the technology and production process creates challenges for effectively creating full traceability. However, RFID holds tremendous potential for improving processing throughput, which will help make RFIDbased traceability more attractive for adoption by meat processors.

"In vitro" study of the molecular mechanism of the "E.Coli" Hsp 70/Hsp40/NEF chaperone system and its interactions with ?-sinuclein

SUMMARY Under stressful conditions, mutant or post-translationally modified proteins may spontaneously misfold and form toxie species, which may further assemble into a continuum of increasingly large and insoluble toxic oligomers that may further condense into less toxic, compact amyloids in the cell Intracellular accumulation of aggregated proteins is a common denominator of several neurodegenerative diseases. To cope with the cytotoxicity induced by abnormal, aggregated proteins, cells have evolved various defence mechanisms among which, the molecular chaperones Hsp70. Hsp70 (DnaK in E. coii) is an ATPase chaperone involved in many physiological processes in the cell, such as assisting de novo protein folding, dissociating native protein oligomers and serving as pulling motors in the import of polypeptides into organelles. In addition, Hsp70 chaperones can actively solubilize and reactivate stable protein aggregates, such as heat- or mutation-induced aggregates. Hsp70 requires the cooperation of two other co-chaperones: Hsp40 and NEF (Nucleotide exchange factor) to fulfil its unfolding activity. In the first experimental section of this thesis (Chapter II), we studied by biochemical analysis the in vitro interaction between recombinant human aggregated α-synuclein (a-Syn oligomers) mimicking toxic a-Syn oligomers species in PD brains, with a model Hsp70/Hsp40 chaperone system (the E. coii DnaK/DnaJ/GrpE). We found that chaperone-mediated unfolding of two denatured model enzymes were strongly affected by α-Syn oligomers but, remarkably, not by monomers. This in vitro observed dysfunction of the Hsp70 chaperone system resulted from the sequestration of the Hsp40 proteins by the oligomeric α-synuclein species. In the second experimental part (Chapter III), we performed in vitro biochemical analysis of the co-chaperone function of three E. coii Hsp40s proteins (DnaJ, CbpA and DjlA) in the ATP-fuelled DnaK-mediated refolding of a model DnaK chaperone substrate into its native state. Hsp40s activities were compared using dose-response approaches in two types of in vitro assays: refolding of heat-denatured G6PDH and DnaK-mediated ATPase activity. We also observed that the disaggregation efficiency of Hsp70 does not directly correlate with Hsp40 binding affinity. Besides, we found that these E. coii Hsp40s confer substrate specificity to DnaK, CbpA being more effective in the DnaK-mediated disaggregation of large G6PDH aggregates than DnaJ under certain conditions. Sensibilisées par différents stress ou mutations, certaines protéines fonctionnelles de la cellule peuvent spontanément se convertir en formes inactives, mal pliées, enrichies en feuillets bêta, et exposant des surfaces hydrophobes favorisant l'agrégation. Cherchant à se stabiliser, les surfaces hydrophobes peuvent s'associer aux régions hydrophobes d'autres protéines mal pliées, formant des agrégats protéiques stables: les amyloïdes. Le dépôt intracellulaire de protéines agrégées est un dénominateur commun à de nombreuses maladies neurodégénératives. Afin de contrer la cytotoxicité induite par les protéines agrégées, les cellules ont développé plusieurs mécanismes de défense, parmi lesquels, les chaperonnes moléculaires Hsp70. Hsp70 nécessite la collaboration de deux autres co-chaperonnes : Hsp40 et NEF pour accomplir son activité de désagrégation. Hsp70 (DnaK, chez E. coli) est impliquée par ailleurs dans d'autres fonctions physiologiques telles que l'assistanat de protéines néosynthétisées à la sortie du ribosome, ou le transport transmembranaire de polypeptides. Par ailleurs, les chaperonnes Hsp70 peuvent également solubiliser et réactiver des protéines agrégées à la suite d'un stress ou d'une mutation. Dans la première partie expérimentale de cette thèse (Chapter II), nous avons étudié in vitro l'interaction entre les oligomères d'a-synucleine, responsables entre autres, de la maladie de Parkinson, et le système chaperon Hsp70/Hsp40 (système Escherichia coli DnaK/DnaJ/GrpE). Nous avons démontré que contrairement aux monomères, les oligomères d'a-synucleine inhibaient le système chaperon lors du repliement de protéines agrégées. Cette dysfonction du système chaperon résulte de la séquestration des chaperonnes Hsp40 par les oligomères d'a-synucleine. La deuxième partie expérimentale (Chapitre III) est consacrée à une étude in vitro de la fonction co-chaperonne de trois Hsp40 d'is. coli (DnaJ, CbpA, et DjlA) lors de la désagrégation par DnaK d'une protéine pré-agrégée. Leurs activités ont été comparées par le biais d'une approche dose-réponse au niveau de deux analyses enzymatiques: le repliement de la protéine agrégée et l'activité ATPase de DnaK. Par ailleurs, nous avons mis en évidence que l'efficacité de désagrégation d'Hsp70 et l'affinité des chaperonnes Hsp40 vis-à-vis de leur substrat n'étaient pas corrélées positivement. Nous avons également montré que ces trois chaperonnes Hsp40 étaient directement impliquées dans la spécificité des fonctions accomplies par les chaperonnes Hsp70. En effet, DnaK en présence de CbpA assure la désagrégation de large agrégats protéiques avec une efficacité nettement plus accrue qu'en présence de DnaJ.