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Find video protocols related to scientific articles indexed in Pubmed.
Nutritional risk among surgery patients and associations with hospital stay and postoperative complications.
Nutr Hosp
PUBLISHED: 09-21-2014
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Nutritional Risk Screening (NRS 2002) is employed to identify nutritional risk in the hospital setting and determine which patients would benefit from nutritional support. The aim of the present study was to identify nutritional risk in patients admitted to the surgery ward and determine possible associations with hospital stay and postoperative complications.
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Mannoprotein MP84 mediates the adhesion of Cryptococcus neoformans to epithelial lung cells.
Front Cell Infect Microbiol
PUBLISHED: 08-19-2014
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The capsule is the most important virulence factor of the fungal pathogen Cryptococcus neoformans. This structure consists of highly hydrated polysaccharides, including glucuronoxylomannan (GXM), and galactoxylomannan (GalXM). It is also composed of mannoproteins (MPs) which corresponds to less than 1% of the capsular weight. Despite MPs being the minority and least studied components, four of these molecules with molecular masses of 115, 98, 88, and 84 kDa were identified and characterized as C. neoformans immunoreactive antigens involved in the pathogenesis, and are potential cryptococcosis vaccine candidates. With the aim to describe the adhesive property of MPs, we cloned and expressed the MP84, a mannoprotein with molecular weight of 84 kDa, on Pichia pastoris yeast, and performed interaction assays of C. neoformans with epithelial lung cells, in the presence or absence of capsule components. Two fungal strains, the wild type, NE-241, and a mutant, CAP67, deficient in GXM production, were used throughout this study. The adhesion assays were completed using epithelial lung cells, A549, and human prostate cancer cells, PC3, as a control. We observed that capsulated wild type (NE-241), and acapsular (CAP67) strains adhered significantly to A549 cells, compared with PC3 cells (p < 0.05). GXM inhibits the NE-241 adhesion, but not the CAP67. In contrast, CAP67 adhesion was only inhibited in the presence of MP84. These results demonstrate the involvement of MP in the adhesion of C. neoformans to epithelial lung cells. We conclude that this interaction possibly involves an adhesion-like interaction between MP on the fungal surface and the complementary receptor molecules on the epithelial cells.
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[Significances of the life experience for the long-lived elderly person in the process of death/dying and mourning].
Cien Saude Colet
PUBLISHED: 08-15-2014
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This is a study using the Heideggerian theoretical-phenomenological approach, which sought to understand the significances of the life experience for the long-lived elderly person in the process of death/dying and mourning. It was conducted in 2009 with 16 long-lived senior citizens of both genders who were aged between 80 and 90, members of a community center for the elderly located in a suburban neighborhood of the city of Salvador in the state of Bahia, Brazil. The results showed that the long-lived elderly person experiences the mourning status process when relatives and friends become ill and die. Furthermore, they gave ambiguous reports with respect to the fear of death. With the attributed significances, it was possible to arrive at the unit of significance, namely the authenticity and lack of authenticity of the individual regarding imminent death. The conclusion reached is that long-lived elderly individuals faced with the process of death/dying and mourning is apparent or concealed in accordance with the moment they are experiencing and the opportunities that present themselves, in other words, it is greatly influenced by their past.
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Identification and functional analysis of Trypanosoma cruzi genes that encode proteins of the glycosylphosphatidylinositol biosynthetic pathway.
PLoS Negl Trop Dis
PUBLISHED: 08-01-2013
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Trypanosoma cruzi is a protist parasite that causes Chagas disease. Several proteins that are essential for parasite virulence and involved in host immune responses are anchored to the membrane through glycosylphosphatidylinositol (GPI) molecules. In addition, T. cruzi GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, T. cruzi genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease.
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Draft Genome Sequence of the Rice Endophyte Burkholderia kururiensis M130.
Genome Announc
PUBLISHED: 04-06-2013
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Burkholderia kururiensis M130 is one of the few characterized rice endophytes and was isolated from surface-sterilized rice roots. This bacterium shows strong growth-promoting effects, being able to increase rice yields. Here we present its draft genome sequence, which contains important traits for endophytic life and plant growth promotion.
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Design, synthesis and trypanocidal evaluation of novel 1,2,4-triazoles-3-thiones derived from natural piperine.
Molecules
PUBLISHED: 03-29-2013
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The work reported herein describes the synthesis and the assessment of the trypanocidal activity of thirteen new 1,2,4-triazole-3-thiones obtained from natural piperine, the main constituent of the dry fruits of Piper nigrum. It is part of a research program aiming to use abundant and easily available natural products as starting materials for the design and synthesis of new molecules potentially useful as antiparasitic drugs. The variously substituted triazole derivatives were synthesized from the natural amide in four steps with the use of microwave irradiation on overall yields ranging from 32% to 51%. The cyclohexyl substituted derivative showed the best trypanocidal profile on proliferative forms of Trypanosoma cruzi (Y strain), with IC??s = 18.3 and 8.87 mM against epimastigotes and amastigotes, respectively.
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Oral motor movements and swallowing in patients with myotonic dystrophy type 1.
Dysphagia
PUBLISHED: 02-08-2013
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Oropharyngeal dysphagia and esophageal motility disorders were found to be the most important causes of aspiration pneumonia in patients with myotonic dystrophy. The purpose of this report was to evaluate clinical characteristics of the oral motor movements and swallowing of individuals with myotonic dystrophy type 1 (DM1) using a standardized clinical protocol and surface electromyography (sEMG). Participants were 40 individuals divided in two groups: G1 composed of 20 adults with DM1 and G2 composed of 20 healthy volunteers paired by age and gender to the individuals in G1. Statistical analysis included one-way ANOVA with two factors for within- and between-group comparisons and Bonferroni correction for multiple comparisons. Patients with DM1 presented deficits in posture, position, and mobility of the oral motor structures, as well as compromised mastication and deglutition. The sEMG data indicated that these patients had longer muscle activations during swallowing events. The longer duration of sEMG in the group of patients with DM1 is possibly related to myotonia and/or incoordination of the muscles involved in the swallowing process or could reflect a physiological adaptation for safe swallowing.
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Addition of ?-O-GlcNAc to threonine residues define the post-translational modification of mucin-like molecules in Trypanosoma cruzi.
Glycoconj. J.
PUBLISHED: 02-06-2013
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Trypanosoma cruzi, an intracellular protozoan etiologic agent of Chagas disease is covered by a dense coat of mucin-type glycoproteins, which is important to promote the parasite entry and persistence in the mammalian host cells. The O-glycosylation of T. cruzi mucins (Tc-mucins) is initiated by enzymatic addition of ?-O-N-acetylglucosamine (GlcNAc) to threonine (Thr) by the UDP-GlcNAc:polypeptide ?-N-acetylglucosaminyltransferase (pp-?-GlcNAcT) in the Golgi. The Tc-mucin is characterized by the presence of a high structural diversity of O-linked oligosaccharides found among different parasite strains, comprising two O-glycan Cores. In the Core 1, from strains principally associated with the domestic transmission cycle of Chagas disease, the GlcNAc O-4 is substituted with a ?-galactopyranose (?Galp) unit, and in the most complex oligosaccharides the GlcNAc O-6 is further processed by the addition of ?1???2-linked Galp residues creating a short linear Galp-containing chain. In the Core 2 structures, expressed by strains isolated from T. cruzi sylvatic hosts, the GlcNAc O-4 carries a ?-galactofuranose (?Galf) unit and the GlcNAc O-6 can carry a branched Galp?1???3[Galp?1???2]Galp?1???6 motif. The O-glycans carrying nonreducing terminal ?Galp are available for sialylation by a surface T. cruzi trans-sialidase activity. Based on structural results, this review summarizes available data on the highly conserved process, which adds the GlcNAc unit in ?-linkage to Thr residues the basis of the post-translational modification system in T. cruzi mucins. In addition, a mechanism unique employed by the parasite to transfer exogenous sialic acid residues to Tc-mucins is presented.
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[Voluntary abortion and domestic violence among women attended at a public maternity hospital of Salvador-BA].
Rev Bras Enferm
PUBLISHED: 12-28-2011
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Quantitative study in order to study domestic violence in women with induced abortion. Interviews were conducted with 147 women hospitalized for induced abortion in a public maternity hospital in Salvador, Bahia. The subjects are characterized by mostly women, black, poorly educated, economically dependent on spouses, experienced psychological abuse, physical and sexual abuse committed by their spouses. Almost half of the women were victims of domestic violence during the current pregnancy, and that was the reason for inducing abortion for 67% of them. The study reveals an association between experience of domestic violence and induced abortion. As mental health consequences, they developed symptoms of post trauma stress disorder. It is necessary that the health professionals consider the cues to identify domestic violence as a health problem associated with induced abortion, which requires a transformation on the training model, including domestic violence as a health issue.
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Reprint of: stuttering treatment control using P300 event-related potentials.
J Fluency Disord
PUBLISHED: 11-21-2011
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Positron emission tomography studies during speech have indicated a failure to show the normal activation of auditory cortical areas in stuttering individuals. In the present study, P300 event-related potentials were used to investigate possible effects of behavioral treatment on the pattern of signal amplitude and latency between waves. In order to compare variations in P300 measurements, a control group paired by age and gender to the group of stutterers, was included in the study. Findings suggest that the group of stutterers presented a significant decrease in stuttering severity after the fluency treatment program. Regarding P300 measurements, stutterers and their controls presented results within normal limits in all testing situations and no significant statistical variations between pre and post treatment testing. When comparing individual results between the testing situations, stutterers presented a higher average decrease in wave latency for the right ear following treatment. The results are discussed in light of previous P300 event-related potentials and functional imaging studies with stuttering adults.
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Molecular and functional characterization of the ceramide synthase from Trypanosoma cruzi.
Mol. Biochem. Parasitol.
PUBLISHED: 07-11-2011
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In this study, we characterized ceramide synthase (CerS) of the protozoan parasite Trypanosoma cruzi at the molecular and functional levels. TcCerS activity was detected initially in a cell-free system using the microsomal fraction of epimastigote forms of T. cruzi, [(3)H]dihydrosphingosine or [(3)H]sphingosine, and fatty acids or acyl-CoA derivatives as acceptor or donor substrates, respectively. TcCerS utilizes both sphingoid long-chain bases, and its activity is exclusively dependent on acyl-CoAs, with palmitoyl-CoA being preferred. In addition, Fumonisin B(1), a broad and well-known acyl-CoA-dependent CerS inhibitor, blocked the parasites CerS activity. However, unlike observations in fungi, the CerS inhibitors Australifungin and Fumonisin B(1) did not affect the proliferation of epimastigotes in culture, even after exposure to high concentrations or after extended periods of treatment. A search of the parasite genome with the conserved Lag1 motif from Lag1p, the yeast acyl-CoA-dependent CerS, identified a T. cruzi candidate gene (TcCERS1) that putatively encodes the parasites CerS activity. The TcCERS1 gene was able to functionally complement the lethality of a lag1? lac1? double deletion yeast mutant in which the acyl-CoA-dependent CerS is not detectable. The complemented strain was capable of synthesizing normal inositol-containing sphingolipids and is 10 times more sensitive to Fumonisin B(1) than the parental strain.
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Stuttering treatment control using P300 event-related potentials.
J Fluency Disord
PUBLISHED: 04-18-2011
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Positron emission tomography studies during speech have indicated a failure to show the normal activation of auditory cortical areas in stuttering individuals. In the present study, P300 event-related potentials were used to investigate possible effects of behavioral treatment on the pattern of signal amplitude and latency between waves. In order to compare variations in P300 measurements, a control group paired by age and gender to the group of stutterers, was included in the study. Findings suggest that the group of stutterers presented a significant decrease in stuttering severity after the fluency treatment program. Regarding P300 measurements, stutterers and their controls presented results within normal limits in all testing situations and no significant statistical variations between pre and post treatment testing. When comparing individual results between the testing situations, stutterers presented a higher average decrease in wave latency for the right ear following treatment. The results are discussed in light of previous P300 event-related potentials and functional imaging studies with stuttering adults. Educational objectives: The reader will learn about and be able to describe the: (1) use of P300 event-related potentials in the study of stuttering; (2) differences between stuttering and non-stuttering adults; and (3) effects of behavioral fluency treatment on cerebral activity in stuttering speakers.
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Common features of environmental and potentially beneficial plant-associated Burkholderia.
Microb. Ecol.
PUBLISHED: 03-28-2011
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The genus Burkholderia comprises more than 60 species isolated from a wide range of niches. Although they have been shown to be diverse and ubiquitously distributed, most studies have thus far focused on the pathogenic species due to their clinical importance. However, the increasing number of recently described Burkholderia species associated with plants or with the environment has highlighted the division of the genus into two main clusters, as suggested by phylogenetical analyses. The first cluster includes human, animal, and plant pathogens, such as Burkholderia glumae, Burkholderia pseudomallei, and Burkholderia mallei, as well as the 17 defined species of the Burkholderia cepacia complex, while the other, more recently established cluster comprises more than 30 non-pathogenic species, which in most cases have been found to be associated with plants, and thus might be considered to be potentially beneficial. Several species from the latter group share characteristics that are of use when associating with plants, such as a quorum sensing system, the presence of nitrogen fixation and/or nodulation genes, and the ability to degrade aromatic compounds. This review examines the commonalities in this growing subgroup of Burkholderia species and discusses their prospective biotechnological applications.
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[Conception of body in Merleau-Ponty and women who were mastectomized].
Rev Bras Enferm
PUBLISHED: 10-25-2010
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Breast cancer has been the responsible for the largest mortality rate in the world, becoming one of the great concerns in health, regarding womans health. Related to the therapeutics, although wide approaches of interventions that make possible better cure perspectives exist, the mastectomy is as treatment base. Article of philosophic reflection, had as study womens that lived the mastectomy aiming at to reflect the conception of these womens body starting from the work Phenomenology of the Perception of Merleau-Ponty object and of authors that work with the theme regarding the woman mastectomized.
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Overlooked post-translational modifications of proteins in Plasmodium falciparum: N- and O-glycosylation -- a review.
Mem. Inst. Oswaldo Cruz
PUBLISHED: 09-25-2010
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Human malignant malaria is caused by Plasmodium falciparum and accounts for almost 900,000 deaths per year, the majority of which are children and pregnant women in developing countries. There has been significant effort to understand the biology of P. falciparum and its interactions with the host. However, these studies are hindered because several aspects of parasite biology remain controversial, such as N- and O-glycosylation. This review describes work that has been done to elucidate protein glycosylation in P. falciparum and it focuses on describing biochemical evidence for N- and O-glycosylation. Although there has been significant work in this field, these aspects of parasite biochemistry need to be explored further.
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Lipopolysaccharide as an antigen target for the formulation of a universal vaccine against Escherichia coli O111 strains.
Clin. Vaccine Immunol.
PUBLISHED: 09-22-2010
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A promising approach to developing a vaccine against O111 strains of diarrheagenic Escherichia coli that exhibit different mechanisms of virulence is to target either the core or the polysaccharide chain (O antigen) of their lipopolysaccharide (LPS). However, due to structural variations found in both these LPS components, to use them as antigen targets for vaccination, it is necessary to formulate a vaccine able to induce a humoral immune response that can recognize all different variants found in E. coli O111 strains. In this study, it was demonstrated that, despite differences in composition of oligosaccharide repeat units between O111ab and O111ac LPS subtypes, antibodies against one O111 subtype can recognize and inhibit the adhesion to human epithelial cells of all categories of O111 E. coli (enteropathogenic E. coli [EPEC], enterohemorrhagic E. coli [EHEC], and enteroaggregative E. coli [EAEC]) strains regardless of the nature of their flagellar antigens, mechanisms of virulence, or O111 polysaccharide subtypes. These antibodies were also able to increase the clearance of different strains of O111 E. coli by macrophages. PCR analyses of the pathways involved in O111 LPS core biosynthesis showed that all EAEC strains have core type R2, whereas typical EPEC and EHEC have core type R3. In contrast, atypical EPEC strains have core types R2 and R3. In summary, the results presented herein indicate that the O111 polysaccharide and LPS core types R2 and R3 are antigen targets for panspecific immunotherapy against all categories of O111 E. coli.
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[Home care to the elderly who had stroke].
Rev Bras Enferm
PUBLISHED: 07-10-2010
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The purpose was to Identify the knowledge production about the stroke in elderly under home care. Bibliographic research whose data were collected though the abstracts from 1997 to 2007, contained in LILACS and SciELO databases. The following key words were used: home assistance, aged people and cerebrovascular accident. Fifty-two references were found in the LILACS database, nine in the SciELO Brazil, and three in the SciELO Cuba. Most of the researches were carried out in 2000. Regarding the method, qualitative method predominance were observed, and central theme is related to the care giver, as well as to the clinical and epidemiologic aspects of the disease. It was observed that this knowledge is still established in Brazil, and the themes related to the person submitted to home care and violence to the aged are still little explored.
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Commonalities and differences in regulation of N-acyl homoserine lactone quorum sensing in the beneficial plant-associated burkholderia species cluster.
Appl. Environ. Microbiol.
PUBLISHED: 04-30-2010
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The genus Burkholderia includes over 60 species isolated from a wide range of environmental niches and can be tentatively divided into two major species clusters. The first cluster includes pathogens such as Burkholderia glumae, B. pseudomallei, and B. mallei and 17 well-studied species of the Burkholderia cepacia complex. The other recently established cluster comprises at least 29 nonpathogenic species, which in most cases have been found to be associated with plants. It was previously established that Burkholderia kururiensis, a member of the latter cluster, possesses an N-acyl homoserine lactone (AHL) quorum-sensing (QS) system designated "BraI/R," which is found in all species of the plant-associated cluster. In the present study, two other BraI/R-like systems were characterized in B. xenovorans and B. unamae and were designated the BraI/R(XEN) and BraI/R(UNA) systems, respectively. Several phenotypes were analyzed, and it was determined that exopolysaccharide was positively regulated by the BraIR-like system in the species B. kururiensis, B. unamae, and B. xenovorans, highlighting commonality in targets. However, the three BraIR-like systems also revealed differences in targets since biofilm formation and plant colonization were differentially regulated. In addition, a second AHL QS system designated XenI2/R2 and an unpaired LuxR solo protein designated BxeR solo were also identified and characterized in B. xenovorans LB400(T). The two AHL QS systems of B. xenovorans are not transcriptionally regulating each other, whereas BxeR solo negatively regulated xenI2. The XenI2/R2 and BxeR solo proteins are not widespread in the Burkholderia species cluster. In conclusion, the present study represents an extensive analysis of AHL QS in the Burkholderia plant-associated cluster demonstrating both commonalities and differences, probably reflecting environmental adaptations of the various species.
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A new class of mechanism-based inhibitors for Trypanosoma cruzi trans-sialidase and their influence on parasite virulence.
Glycobiology
PUBLISHED: 04-22-2010
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One of the most interesting aspects of Trypanosoma cruzi is its adaptation to obtain sialic acid from its host, fulfilling this need exclusively through the reaction catalyzed by enzymatically active trans-sialidase (aTS), thought to play an important role in the pathogenesis of Chagas disease. Herein, we report that 2-difluoromethyl-4-nitrophenyl-3,5-dideoxy-d-glycero-alpha-d-galacto-2-nonulopyranosid acid (NeuNAcFNP) inactivates aTS time- and dose-dependently, and this inhibition was not relieved removing the inhibitor. Also, NeuNAcFNP causes a decrease in infection of mammalian cells. Characterization of labeled aTS by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed that inactivation of the enzyme occurs through formation of a covalent bond between Arg245 and Asp247 and the inhibitor aglycone. Participation of Asp247 in the catalytic mechanism was proved by constructing a TSD247A mutant, which presents only residual activity. Molecular dynamic simulations indicate that the D247A mutation results in a more open catalytic cleft. In summary, NeuNAcFNP is the first reported mechanism-based inhibitor of aTS, representing a new template for drug design and opening new possibilities for chemotherapy of Chagas disease, as well as for the elucidation of aTS function in T. cruzi pathogenesis and biology.
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Trypanosoma cruzi subverts host cell sialylation and may compromise antigen-specific CD8+ T cell responses.
J. Biol. Chem.
PUBLISHED: 01-27-2010
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Upon activation, cytotoxic CD8(+) T lymphocytes are desialylated exposing beta-galactose residues in a physiological change that enhances their effector activity and that can be monitored on the basis of increased binding of the lectin peanut agglutinin. Herein, we investigated the impact of sialylation mediated by trans-sialidase, a specific and unique Trypanosoma transglycosylase for sialic acid, on CD8(+) T cell response of mice infected with T. cruzi. Our data demonstrate that T. cruzi uses its trans-sialidase enzyme to resialylate the CD8(+) T cell surface, thereby dampening antigen-specific CD8(+) T cell response that might favor its own persistence in the mammalian host. Binding of the monoclonal antibody S7, which recognizes sialic acid-containing epitopes on the 115-kDa isoform of CD43, was augmented on CD8(+) T cells from ST3Gal-I-deficient infected mice, indicating that CD43 is one sialic acid acceptor for trans-sialidase activity on the CD8(+) T cell surface. The cytotoxic activity of antigen-experienced CD8(+) T cells against the immunodominant trans-sialidase synthetic peptide IYNVGQVSI was decreased following active trans-sialidase-mediated resialylation in vitro and in vivo. Inhibition of the parasites native trans-sialidase activity during infection strongly decreased CD8(+) T cell sialylation, reverting it to the glycosylation status expected in the absence of parasite manipulation increasing mouse survival. Taken together, these results demonstrate, for the first time, that T. cruzi subverts sialylation to attenuate CD8(+) T cell interactions with peptide-major histocompatibility complex class I complexes. CD8(+) T cell resialylation may represent a sophisticated strategy to ensure lifetime host parasitism.
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Growing self-reconstruction maps.
IEEE Trans Neural Netw
PUBLISHED: 12-11-2009
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In this paper, we propose a new method for surface reconstruction based on growing self-organizing maps (SOMs), called growing self-reconstruction maps (GSRMs). GSRM is an extension of growing neural gas (GNG) that includes the concept of triangular faces in the learning algorithm and additional conditions in order to include and remove connections, so that it can produce a triangular two-manifold mesh representation of a target object given an unstructured point cloud of its surface. The main modifications concern competitive Hebbian learning (CHL), the vertex insertion operation, and the edge removal mechanism. The method proposed is able to learn the geometry and topology of the surface represented in the point cloud and to generate meshes with different resolutions. Experimental results show that the proposed method can produce models that approximate the shape of an object, including its concave regions, boundaries, and holes, if any.
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Structural elucidation of the repeat unit in highly branched acidic exopolysaccharides produced by nitrogen fixing Burkholderia.
Glycobiology
PUBLISHED: 11-18-2009
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Burkholderia kururiensis, strain M130, an endophytic diazotrophic bacterium isolated from rice roots, produces acetylated acidic exopolysaccharides which can be separated by anion exchange chromatography. These were characterized by nuclear magnetic resonance spectroscopy, methylation analysis and Smith degradation. The exopolysaccharides eluted with 0.5 M NaCl were produced when the bacterium was grown in a medium containing mannitol as the sole carbon source, and showed to be a mixture of two different polymers, composed of hepta or octasaccharide repeat units, consistent with following structure: [structure: see text]. The ability of diazotrophic Burkholderia to produce two exopolysaccharides that differ by the presence of a terminal glucosyl residue provides insight into polysaccharide function with potentially significant biological consequences in the endophytic-host plant interaction.
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The major surface carbohydrates of the Echinococcus granulosus cyst: mucin-type O-glycans decorated by novel galactose-based structures.
Biochemistry
PUBLISHED: 11-03-2009
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The cestodes constitute important but understudied human and veterinary parasites. Their surfaces are rich in carbohydrates, on which very little structural information is available. The tissue-dwelling larva (hydatid cyst) of the cestode Echinococcus granulosus is outwardly protected by a massive layer of carbohydrate-rich extracellular matrix, termed the laminated layer. The monosaccharide composition of this layer suggests that its major carbohydrate components are exclusively mucin-type O-glycans. We have purified these glycans after their release from the crude laminated layer and obtained by MS and NMR the complete structure of 10 of the most abundant components. The structures, between two and six residues in length, encompass a limited number of biosynthetic motifs. The mucin cores 1 and 2 are either nondecorated or elongated by a chain of Galpbeta1-3 residues. This chain can be capped by a single Galpalpha1-4 residue, such capping becoming more dominant with increasing chain size. In addition, the core 2 N-acetylglucosamine residue is in cases substituted with the disaccharide Galpalpha1-4Galpbeta1-4, giving rise to the blood P(1)-antigen motif. Larger, also related, glycans exist, reaching at least 18 residues in size. The glycans described are related but larger than those previously described from an Echinococcus multilocularis mucin [Hulsmeier, A. J., et al. (2002) J. Biol. Chem. 277, 5742-5748]. Our results reveal that the E. granulosus cyst exposes to the host only a few different major carbohydrate motifs. These motifs are composed essentially of galactose units and include the elongation by (Galpbeta1-3)(n) and the capping by Galpalpha1-4, novel in animal mucin-type O-glycans.
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Sorting of phosphoglucomutase to glycosomes in Trypanosoma cruzi is mediated by an internal domain.
Glycobiology
PUBLISHED: 08-20-2009
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Trypanosoma cruzi relies on highly galactosylated molecules as virulence factors and the enzymes involved in sugar biosynthesis are potential therapeutic targets. The synthesis of UDP-galactose in T. cruzi requires the activity of phosphoglucomutase (PGM), the enzyme that catalyzes the interconversion of glucose-6-phosphate and glucose-1-phosphate. Several enzymes that participate in carbohydrate metabolism in trypanosomes are confined to specialized peroxisome-like organelles called glycosomes. The majority of glycosomal proteins contain peroxisome-targeting signals (PTS) at the COOH- or at the amino-terminus, which drive their transport to glycosomes. We had previously identified the T. cruzi PGM gene (TcPGM) and demonstrated that it encodes a functional enzyme. Here, we show that, in contrast to yeast and mammalian cells, TcPGM resides in glycosomes of the parasite. However, no classical PTS1 or PTS2 motif is present in its sequence. We investigated glycosomal targeting by generating T. cruzi cell lines expressing different domains of TcPGM fused to the green fluorescent protein (GFP). The analysis of the subcellular localization of fusion proteins revealed that an internal targeting signal of TcPGM, residing between amino acid residues 260 and 380, is capable of targeting GFP to glycosomes. These results demonstrate that, in T. cruzi, PGM import into glycosomes is mediated by a novel non-PTS domain that is located internally in the protein.
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Molecular analysis of a UDP-GlcNAc:polypeptide alpha-N-acetylglucosaminyltransferase implicated in the initiation of mucin-type O-glycosylation in Trypanosoma cruzi.
Glycobiology
PUBLISHED: 05-25-2009
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Trypanosoma cruzi, the causative agent of Chagas disease, is surrounded by a mucin coat that plays important functions in parasite survival/invasion and is extensively O-glycosylated by Golgi and cell surface glycosyltransferases. The addition of the first sugar, alpha-N-acetylglucosamine (GlcNAc) linked to Threonine (Thr), is catalyzed by a polypeptide alpha-GlcNAc-transferase (pp-alphaGlcNAcT) which is unstable to purification. Here, a comparison of the genomes of T. cruzi and Dictyostelium discoideum, an amoebazoan which also forms this linkage, identified two T. cruzi genes (TcOGNT1 and TcOGNT2) that might encode this activity. Though neither was able to complement the Dictyostelium gene, expression in the trypanosomatid Leishmania tarentolae resulted in elevated levels of UDP-[(3)H]GlcNAc:Thr-peptide GlcNAc-transferase activity and UDP-[(3)H]GlcNAc breakdown activity. The ectodomain of TcOGNT2 was expressed and the secreted protein was found to retain both activities after extensive purification away from other proteins and the endogenous activity. Product analysis showed that (3)H was transferred as GlcNAc to a hydroxyamino acid, and breakdown was due to hydrolysis. Both activities were specific for UDP-GlcNAc relative to UDP-GalNAc and were abolished by active site point mutations that inactivate a related Dictyostelium enzyme and distantly related animal pp-alphaGalNAcTs. The peptide preference and the alkaline pH optimum were indistinguishable from those of the native activity in T. cruzi microsomes. The results suggest that mucin-type O-glycosylation in T. cruzi is initiated by conserved members of CAZy family GT60, which is homologous to the GT27 family of animal pp-alphaGalNAcTs that initiate mucin-type O-glycosylation in animals.
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Alpha-N-acetylglucosamine-linked O-glycans of sialoglycoproteins (Tc-mucins) from Trypanosoma cruzi Colombiana strain.
Mem. Inst. Oswaldo Cruz
PUBLISHED: 05-12-2009
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Trypanosoma cruzi sialoglycoproteins (Tc-mucins) are mucin-like molecules linked to a parasite membrane via a glycosylphosphatidylinositol anchor. We previously determined the structures of Tc-mucin O-glycan domains from several T. cruzi strains and observed significant differences among them. We now report the amino acid content and structure of Tc-mucin O-glycan chains from T. cruzi Colombiana, a strain resistant to common trypanocidal drugs. Amino acid analysis demonstrated the predominance of threonine residues (42%) and helped to identify the O-glycans as belonging to a Tc-mucin family that contain a beta-galactofuranose (beta-Galf) residue attached to an alpha-N-acetylglucosamine (alpha-GlcNAc) O-4, with the most complex glycan, a pentasaccharide-GlcNAc-ol with a branched trigalactopyranose chain, on the GlcNAc O-6. The presence of beta-Galf on O-glycans from T. cruzi Colombiana mucins supports the use of glycosylation as a phylogenetic marker for the classification of Colombiana in the T. cruzi I group.
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Further structural characterization of the Echinococcus granulosus laminated layer carbohydrates: the blood-antigen P1-motif gives rise to branches at different points of the O-glycan chains.
Glycobiology
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The glycobiology of the cestodes, a class of parasitic flatworms, is still largely unexplored. An important cestode species is Echinococcus granulosus, the tissue-dwelling larval stage of which causes hydatid disease. The E. granulosus larva is protected from the host by a massive mucin-based extracellular matrix termed laminated layer (LL). We previously reported ( Díaz et al. 2009. Biochemistry 48:11678-11691) the molecular structure of the most abundant LL O-glycans, comprising up to six monosaccharide residues. These are based on Cores 1 and 2, in cases elongated by a chain of Galp?1-3 residues, which can be capped by Galp?1-4. In addition, the Core 2 GlcNAcp residue can be decorated with the Galp?1-4Galp?1-4 disaccharide. Larger glycans also detected contained additional HexNAc residues that could not be explained by the structural repertoire described above. In this work, we elucidate, by mass spectrometry (MS) and nuclear magnetic resonance (NMR), six additional glycans from the E. granulosus LL between six and eight residues in size. Their structures are related to those already described but in cases bear GlcNAcp?1-6 or Galp?1-4Galp?1-4GlcNAcp?1-6 as ramifications on the core Galp?1-3 residue. We also obtained evidence that noncore Galp?1-3 residues can be similarly ramified. Thus, the new motif together with the previous information may explain all the glycan compositions detected in the LL by MS. In addition, we show that the anti-Echinococcus monoclonal antibody E492 (Parasite Immunol 21:141, 1999) recognizes Galp?1-4Galp?1-4GlcNAcp (the blood P(1)-antigen motif). This explains the antibodys reactivity with a range of Echinococcus tissues, as the P(1)-motif is also carried on non-LL N-glycans and glycolipids from this genus.
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Glycoinositolphospholipids from Trypanosomatids subvert nitric oxide production in Rhodnius prolixus salivary glands.
PLoS ONE
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Rhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands.
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Trypanosoma cruzi adjuvants potentiate T cell-mediated immunity induced by a NY-ESO-1 based antitumor vaccine.
PLoS ONE
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Immunological adjuvants that induce T cell-mediate immunity (TCMI) with the least side effects are needed for the development of human vaccines. Glycoinositolphospholipids (GIPL) and CpGs oligodeoxynucleotides (CpG ODNs) derived from the protozoa parasite Trypanosoma cruzi induce potent pro-inflammatory reaction through activation of Toll-Like Receptor (TLR)4 and TLR9, respectively. Here, using mouse models, we tested the T. cruzi derived TLR agonists as immunological adjuvants in an antitumor vaccine. For comparison, we used well-established TLR agonists, such as the bacterial derived monophosphoryl lipid A (MPL), lipopeptide (Pam3Cys), and CpG ODN. All tested TLR agonists were comparable to induce antibody responses, whereas significant differences were noticed in their ability to elicit CD4(+) T and CD8(+) T cell responses. In particular, both GIPLs (GTH, and GY) and CpG ODNs (B344, B297 and B128) derived from T. cruzi elicited interferon-gamma (IFN-?) production by CD4(+) T cells. On the other hand, the parasite derived CpG ODNs, but not GIPLs, elicited a potent IFN-? response by CD8(+) T lymphocytes. The side effects were also evaluated by local pain (hypernociception). The intensity of hypernociception induced by vaccination was alleviated by administration of an analgesic drug without affecting protective immunity. Finally, the level of protective immunity against the NY-ESO-1 expressing melanoma was associated with the magnitude of both CD4(+) T and CD8(+) T cell responses elicited by a specific immunological adjuvant.
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