Application of WHO International Biological Reference Standards to evaluate commercial serological tests for chronic Chagas disease.

BACKGROUND Chagas disease, resulting from Trypanosoma cruzi infections, continues to be a health concern mainly in Latin American countries where the parasite is endemic. The laboratory diagnosis of a chronic infection is determined through serological assays for antibodies against T. cruzi and several tests are available that differ in key components, formats and methodologies. To date, no single test meets the criteria of a gold standard. The situation is further complicated by the difficulties associated with performance comparisons between different immunoassays or methodologies executed at different times and geographical areas. OBJECTIVE To improve the diagnosis of Chagas disease, the WHO coordinated the development of two International Biological Reference Standards for antibodies against anti-T. cruzi: NIBSC 09/186 and NIBSC 09/188 that respectively represent geographical regions with the highest prevalence of TcII and TcI lineages of the parasite. METHODS The principle goal of this study was to verify the behavior of these standards when assayed by several commercially available serological tests that employ different methods to capture and detect human anti-T. cruzi antibodies. FINDINGS AND MAIN CONCLUSIONS The results reinforce the recommendation that these standards be considered for performance evaluations of commercialised immunoassays and should be an integral step in the development of new test components or assay paradigms.

Physical exercise stimulates salivary secretion of cystatins.

Physical exercise is known to activate the sympathetic nervous system, which influences the production of saliva from salivary glands. Our examination of saliva collected from highly trained athletes before and after a number of physical competititions showed an increase in the secretion of S-type cystatins and cystatin C as a subacute response to aerobic and anaerobic exercise. The elevation in salivary cystatins was transient and the recovery time course differed from that of amylase and other salivary proteins. An in vitro assay was developed based on a cell line from a human submandibular gland (HSG) that differentiated into acinus-like structures. Treatments with the ß-adrenergic agonist isoproterenol caused a shift in the intracellular distribution of S-type cystatins and cystatin C, promoting their accumulation at the outer regions of the acinus prior to release and suggesting the activation of a directional transport involving co-migration of both molecules. In another treatment using non-differentiated HSG cells, it was evident that both expression and secretion of cystatin C increased upon addition of the ß-adrenergic agonist, and these effects were essentially eliminated by the antagonist propranolol. The HSG cell line appears to have potential as a model for exploring the mechanism of cystatin secretion, particularly the S-type cystatins that originate primarily in the submandibular glands.

Separation of Volatile Metabolites from the Leaf-Derived Essential Oil of Piper mollicomum Kunth (Piperaceae) by High-Speed Countercurrent Chromatography.

The technique of high-speed countercurrent chromatography was applied to the isolation of compounds in essential oil derived from the leaves of Piper mollicomum species. Plant leaves (200.0 g) were submitted to hydrodistillation in a modified Clevenger apparatus. The resulting crude leaf essential oil was analyzed by gas chromatography with flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS) to determine the profile of the components. The purified fractions were composed of monoterpenes and sesquiterpenes such as camphor (85.0 mg at 98.5% purity), (E)-nerolidol (100.0 mg at 92.8% purity), and camphene (150.0 mg at 82.0% purity). A minor component of the essential oil, bornyl acetate (16.2 mg at 91.2% purity) was also isolated in the one-step separation protocol in 2 h. The countercurrent chromatography technique proved to be a fast and efficient method for the separation of volatile metabolites that conserved the solvent while delivering various fractions of high purity.

Impairing the function of MLCK, myosin Va or myosin Vb disrupts Rhinovirus B14 replication.

Together, the three human rhinovirus (RV) species are the most frequent cause of the common cold. Because of their high similarity with other viral species of the genus Enterovirus, within the large family Picornaviridae, studies on RV infectious activities often offer a less pathogenic model for more aggressive enteroviruses, e.g. poliovirus or EV71. Picornaviruses enter via receptor mediated endocytosis and replicate in the cytosol. Most of them depend on functional F-actin, Rab proteins, and probably motor proteins. To assess the latter, we evaluated the role of myosin light chain kinase (MLCK) and two myosin V isoforms (Va and Vb) in RV-B14 infection. We report that ML-9, a very specific MLCK inhibitor, dramatically reduced RV-B14 entry. We also demonstrate that RV-B14 infection in cells expressing dominant-negative forms of myosin Va and Vb was impaired after virus entry. Using immunofluorescent localization and immunoprecipitation, we show that myosin Va co-localized with RV-B14 exclusively after viral entry (15 min post infection) and that myosin Vb was present in the clusters of newly synthesized RNA in infected cells. These clusters, observed at 180 min post infection, are reminiscent of replication sites. Taken together, these results identify myosin light chain kinase, myosin Va and myosin Vb as new players in RV-B14 infection that participate directly or indirectly in different stages of the viral cycle.

Echinodorus grandiflorus: Ethnobotanical, phytochemical and pharmacological overview of a medicinal plant used in Brazil.

Echinodorus grandiflorus (Cham. & Schltdl.) Micheli is a native Brazilian species used in traditional practices for the treatment of several conditions such as inflammatory diseases, arthritis and hypertension. Through a systematic review of the accumulated knowledge about the species E. grandiflorus, the botanical, phytochemistry, ethnobotanical and pharmacological properties of this medicinal plant demonstrates its potential to naturally provide anti-inflammatory and anti-oxidant with a special emphasis on anti-hypertensive and cardioprotective effects. The body of literature reports that the chemical composition of crude E. grandiflorus extracts are notably composed of diterpenoids and flavonoids metabolites. Pharmacological studies have shown that oral treatments using the hydroalcoholic extracts of leaves from this plant has a significant anti-inflammatory, anti-hypertensive, diuretic and cardioprotective effects in rats with no toxicity. The holistic activities of complex extracts are corroborated by the individuals mechanisms of action, as well as, synergistic benefits attributed to the isolated chemical major constituents in this species. In light of the serious health concerns ascribed, it is important to investigate medicinal plant species with histories of traditional use for circulatory problems to meet the growing demands by scientifically validating their use and safety.

Exogenous ß-amyloid peptide interferes with GLUT4 localization in neurons.

Aging represents a major risk factor for numerous illnesses that are of increasing importance to society, including two of the most prevalent: diabetes and Alzheimer's disease. Studies have shown that diabetes is a risk factor for spontaneous Alzheimer's disease. While these studies suggest that diabetes can contribute to Alzheimer's disease, the implications of AD on diabetes are practically unexplored. The major mediator of the pathophysiological effects, the Aß42 peptide, has been shown to enter neurons and lead to an alteration of the intracellular distribution of the molecular motor myosin Vb. Myosin Vb functions in memory and learning by participating in the strengthening of the long-term potentiation (LTP) of synaptic transmissions. It has also been implicated in the translocation of the glucose transporter, GLUT4, to the plasma membrane in response to insulin, a process that is defective in diabetes. Here, the effect on GLUT4 upon entry of the Aß42 peptide into cultured chick retinal neurons was explored. The results suggest an alteration in distribution and a reduced level at the cell surface, as well as an increased colocalization with myosin Vb, which can partially explain the changes in glucose metabolism associated with AD. It is also shown that the presence of the Aß40 peptide inhibits the internalization of the Aß42 peptide in cultured cells. Together, the results provide additional targets for the development of therapeutics against the progression and effects of Alzheimer's disease.

Myosin5a tail associates directly with Rab3A-containing compartments in neurons.

Myosin-Va (Myo5a) is a motor protein associated with synaptic vesicles (SVs) but the mechanism by which it interacts has not yet been identified. A potential class of binding partners are Rab GTPases and Rab3A is known to associate with SVs and is involved in SV trafficking. We performed experiments to determine whether Rab3A interacts with Myo5a and whether it is required for transport of neuronal vesicles. In vitro motility assays performed with axoplasm from the squid giant axon showed a requirement for a Rab GTPase in Myo5a-dependent vesicle transport. Furthermore, mouse recombinant Myo5a tail revealed that it associated with Rab3A in rat brain synaptosomal preparations in vitro and the association was confirmed by immunofluorescence imaging of primary neurons isolated from the frontal cortex of mouse brains. Synaptosomal Rab3A was retained on recombinant GST-tagged Myo5a tail affinity columns in a GTP-dependent manner. Finally, the direct interaction of Myo5a and Rab3A was determined by sedimentation velocity analytical ultracentrifugation using recombinant mouse Myo5a tail and human Rab3A. When both proteins were incubated in the presence of 1 mm GTPγS, Myo5a tail and Rab3A formed a complex and a direct interaction was observed. Further analysis revealed that GTP-bound Rab3A interacts with both the monomeric and dimeric species of the Myo5a tail. However, the interaction between Myo5a tail and nucleotide-free Rab3A did not occur. Thus, our results show that Myo5a and Rab3A are direct binding partners and interact on SVs and that the Myo5a/Rab3A complex is involved in transport of neuronal vesicles.

Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity.

Learning-related plasticity at excitatory synapses in the mammalian brain requires the trafficking of AMPA receptors and the growth of dendritic spines. However, the mechanisms that couple plasticity stimuli to the trafficking of postsynaptic cargo are poorly understood. Here we demonstrate that myosin Vb (MyoVb), a Ca2+-sensitive motor, conducts spine trafficking during long-term potentiation (LTP) of synaptic strength. Upon activation of NMDA receptors and corresponding Ca2+ influx, MyoVb associates with recycling endosomes (REs), triggering rapid spine recruitment of endosomes and local exocytosis in spines. Disruption of MyoVb or its interaction with the RE adaptor Rab11-FIP2 abolishes LTP-induced exocytosis from REs and prevents both AMPA receptor insertion and spine growth. Furthermore, induction of tight binding of MyoVb to actin using an acute chemical genetic strategy eradicates LTP in hippocampal slices. Thus, Ca2+-activated MyoVb captures and mobilizes REs for AMPA receptor insertion and spine growth, providing a mechanistic link between the induction and expression of postsynaptic plasticity.

Myosin-Vb functions as a dynamic tether for peripheral endocytic compartments during transferrin trafficking.

BACKGROUND: Myosin-Vb has been shown to be involved in the recycling of diverse proteins in multiple cell types. Studies on transferrin trafficking in HeLa cells using a dominant-negative myosin-Vb tail fragment suggested that myosin-Vb was required for recycling from perinuclear compartments to the plasma membrane. However, chemical-genetic, dominant-negative experiments, in which myosin-Vb was specifically induced to bind to actin, suggested that the initial hypothesis was incorrect both in its site and mode of myosin-Vb action. Instead, the chemical-genetic data suggested that myosin-Vb functions in the actin-rich periphery as a dynamic tether on peripheral endosomes, retarding transferrin transport to perinuclear compartments. RESULTS: In this study, we employed both approaches, with the addition of overexpression of full-length wild-type myosin-Vb and switching the order of myosin-Vb inhibition and transferrin loading, to distinguish between these hypotheses. Overexpression of full-length myosin-Vb produced large peripheral endosomes. Chemical-genetic inhibition of myosin-Vb after loading with transferrin did not prevent movement of transferrin from perinuclear compartments; however, virtually all myosin-Vb-decorated particles, including those moving on microtubules, were halted by the inhibition. Overexpression of the myosin-Vb tail caused a less-peripheral distribution of early endosome antigen-1 (EEA1). CONCLUSION: All results favored the peripheral dynamic tethering hypothesis.

Myosin-Va mediates RNA distribution in primary fibroblasts from multiple organs.

Myosin-Va has been shown to have multiple functions in a variety of cell types, including a role in RNA transport in neurons. Using primary cultures of cells from organs of young dilute-lethal (Myo5a(d-l)/Myo5a(d-l)) null mutant mice and wild-type controls, we show that in some, but not all, tissues, RNA distribution is dramatically different in the homozygous null mutant cells. The dependence of RNA localization on myosin-Va correlates with the relative abundance of the brain-specific splicing pattern of the myosin-Va tail. We also show that myosin-Va is involved in RNA localization soon after synthesis, because the effects of its absence are diminished for RNAs that are more than 30 min old. Finally, we show that localization of beta-actin mRNA is significantly changed by the absence of myosin-Va. These results suggest that myosin-Va is involved in a transient transport or tethering function in the perinuclear region.

Chemical-genetic inhibition of sensitized mutant unconventional myosins.

The construction of a sensitized mutant unconventional myosin is an excellent method for determining the function of the individual myosin against a background of related myosins with partially overlapping functions. In this chapter, we outline the steps involved in sensitizing myosin by mutation and screening them against panels of nucleotide analogs, including transfection, microinjection, and actin co-sedimentation in vitro. We also describe conditions and considerations involved in designing functional experiments after a mutant and cognate analog have been identified. The powerful strategy of chemical genetics, when correctly applied to unconventional myosins, enables both the specific and selectable inhibition of the target motor with outstanding internal controls.

Chemical-genetic inhibition of a sensitized mutant myosin Vb demonstrates a role in peripheral-pericentriolar membrane traffic.

Selective, in situ inhibition of individual unconventional myosins is a powerful approach to determine their specific physiological functions. Here, we report the engineering of a myosin Vb mutant that still hydrolyzes ATP, yet is selectively sensitized to an N(6)-substituted ADP analog that inhibits its activity, causing it to remain tightly bound to actin. Inhibition of the sensitized mutant causes inhibition of accumulation of transferrin in the cytoplasm and increases levels of plasma-membrane transferrin receptor, suggesting that myosin Vb functions in traffic between peripheral and pericentrosomal compartments.

Melanophilin, the product of the leaden locus, is required for targeting of myosin-Va to melanosomes.

The formation of complex subcellular organelles requires the coordinated targeting of multiple components. Melanosome biogenesis in mouse melanocytes is an excellent model system for studying the coordinated function of multiple gene products in intracellular trafficking. To begin to order events in melanosome biogenesis and distribution, we employed the classical coat-color mutants ashen, dilute, and leaden, which affect melanosome distribution, but not melanin synthesis. The loci have been renamed Rab27a, Myo5a, and Mlph for their gene products. While each of the three loci has been shown to be required for melanosome distribution, the point(s) at which each acts is unknown. We have utilized primary melanocytes to examine the interdependencies between rab27a, myosin-Va, and melanophilin. The localization of rab27a to melanosomes did not require the function of either myosin-Va or melanophilin, but leaden function was required for the association of myosin-Va with melanosomes. In leaden melanocytes permeabilized before fixation, myosin-Va immunoreactivity was greatly attenuated, suggesting that myosin-Va is free in the cytoplasm. Finally, we have complemented both the leaden and ashen phenotypes by cell fusion and observed redistribution of mature melanosomes in the absence of both protein and melanin synthesis. Together, our data suggest a model for the initial assembly of the machinery required for melanosome distribution.

A chemical-genetic strategy implicates myosin-1c in adaptation by hair cells.

Myosin-1c (also known as myosin-Ibeta) has been proposed to mediate the slow component of adaptation by hair cells, the sensory cells of the inner ear. To test this hypothesis, we mutated tyrosine-61 of myosin-1c to glycine, conferring susceptibility to inhibition by N(6)-modified ADP analogs. We expressed the mutant myosin-1c in utricular hair cells of transgenic mice, delivered an ADP analog through a whole-cell recording pipette, and found that the analog rapidly blocked adaptation to positive and negative deflections in transgenic cells but not in wild-type cells. The speed and specificity of inhibition suggests that myosin-1c participates in adaptation in hair cells.