isolation-human-mesenchymal-stem-cells-their-cultivation-on-porous

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Universidad Nacional Autónoma de México (UNAM)

Identification, localization and partial biochemical characterization of actins expressed in the larval stage of the cestode parasite Taenia solium has been carried out. Frozen tissue sections of cysticerci, the larval stage of this parasite, were reacted with rhodamine-phalloidin, parasite actin was purified by polymerization in the presence of K(+), Mg(++) and ATP actin was analyzed by SDS-PAGE and two-dimensional gel electrophoresis, and immunoblotting of actin was performed in PVDF membranes and with commercial anti-actin monoclonal antibodies. Parasitic tissues showed different fibrous actin fluorescence patterns, which correlated with the expression of isoactins. Purified globular actin had a similar molecular mass to rabbit commercial actin (approximately 44 kDa). Actin was resolved into seven isoforms, indicating a family of actin genes.

Actin expression in Taenia solium cysticerci (cestoda): tisular distribution and detection of isoforms.

Type II myosin, the primary component of the thick filament of muscle fibers, is organized as a dimeric high molecular weight protein, and is composed of a pair of heavy chains (MHC) and two pairs of light chains. Myosin II transforms ATP energy into mechanical force. All type II myosins are conserved proteins but they have two variable regions that are located in different places of the molecule. Myosin molecules are encoded by a multigene family and many isoforms are generated. The expression of myosins depends on the developmental stage and on the type and degree of contractile activity and tissue, therefore several myosin isoforms are found in the same organism. Here we describe the use of different techniques that allowed demonstrating the presence of isoforms of the heavy chain type II myosin of Taenia solium cysticerci (larvae) and tapeworms (adults), a cestode parasite of importance in public health in many developing countries. Myosin was purified and used in comparative proteolytic fragmentation, ATPase activity, detection of antigenic differences and electrophoretic separation. The results obtained showed biochemical and immunochemical differences among cysticerci and tapeworms, and demonstrate the presence of myosin isoforms in T. solium that are probably associated to physiological requirements of each developmental stage.

Muscular myosin isoforms of Taenia solium (Cestoda).

Using a murine model of cysticercosis caused by the Taenia crassiceps ORF strain, we developed a fluorescent quantitative evaluation of the action of two well known anti-helminthic drugs: albendazole sulfoxide and praziquantel. The fluorescence emitted by a biotransformed CellTracker Probe known as CellTracker Green CMFDA in the vesicular fluids of cysticerci was estimated, and the results were compared with macroscopic observations of the parasites. The pharmacological EC(50) value of each drug and changes in the level of biotransformation of the fluorescent tracker caused by the drugs could be easily calculated. These drug-induced changes in biotransformation could be related to changes in the GSH/GSSG ratio of parasites. Both the cysticercosis murine model and the CMFDA biotransformation assay could be used as an in vitro screening method to evaluate potential or well known cysticidal drugs.

In vitro evaluation of the effects of cysticidal drugs in the Taenia crassiceps cysticerci ORF strain using the fluorescent CellTracker CMFDA.

The expression and biological role of actin during the Trypanosoma cruzi life cycle remains largely unknown. Polyclonal antibodies against a recombinant T. cruzi actin protein were used to confirm its expression in epimastigotes, trypomastigotes, and amastigotes. Although the overall levels of expression were similar, clear differences in the subcellular distribution of actin among the developmental stages were identified. The existence of five actin variants in each developmental stage with distinct patterns of expression were uncovered by immunoblotting of protein extracts separated 2D-SDS gels. The isoelectric points of the actin variants in epimastigotes ranged from 4.45 to 4.9, whereas they ranged from 4.9 to 5.24 in trypomastigotes and amastigotes. To determine if the actin variants found could represent previously unidentified actins, we performed a genomic survey of the T.cruzi GeneDB database and found 12 independent loci encoding for a diverse group of actins and actin-like proteins that are conserved among trypanosomatids.

Trypanosoma cruzi: multiple actin isovariants are observed along different developmental stages.

Flame cells are the terminal cells of protonephridial systems, which are part of the excretory systems of invertebrates. Although the knowledge of their biological role is incomplete, there is a consensus that these cells perform excretion/secretion activities. It has been suggested that the flame cells participate in the maintenance of the osmotic environment that the cestodes require to live inside their hosts. In live Platyhelminthes, by light microscopy, the cells appear beating their flames rapidly and, at the ultrastructural, the cells have a large body enclosing a tuft of cilia. Few studies have been performed to define the localization of the cytoskeletal proteins of these cells, and it is unclear how these proteins are involved in cell function.

Visualization and 3D reconstruction of flame cells of Taenia solium (cestoda).

The role of an estrogen-binding protein similar to a known mammalian estrogen receptor (ER) is described in the estradiol-dependent reproduction of the helminth parasite Taenia crassiceps. Previous results have shown that 17-β-estradiol induces a concentration-dependent increase in bud number of in vitro cultured cysticerci. This effect is inhibited when parasites are also incubated in the presence of an ER binding-inhibitor (tamoxifen). RT-PCR assays using specific oligonucleotides of the most conserved ER sequences, showed expression by the parasite of a mRNA band of molecular weight and sequence corresponding to an ER. Western blot assays revealed reactivity with a 66 kDa protein corresponding to the parasite ER protein. Tamoxifen treatment strongly reduced the production of the T. crassiceps ER-like protein. Antibody specificity was demonstrated by immunoprecipitating the total parasite protein extract with anti-ER-antibodies. Cross-contamination by host cells was discarded by flow cytometry analysis. ER was specifically detected on cells expressing paramyosin, a specific helminth cell marker. Parasite cells expressing the ER-like protein were located by confocal microscopy in the subtegumental tissue exclusively. Analysis of the ER-like protein by bidimensional electrophoresis and immunoblot identified a specific protein of molecular weight and isoelectric point similar to a vertebrates ER. Sequencing of the spot produced a small fragment of protein similar to the mammalian nuclear ER. Together these results show that T. crassiceps expresses an ER-like protein which activates the budding of T. crassiceps cysticerci in vitro. To the best of our knowledge, this is the first report of an ER-like protein in parasites. This finding may have strong implications in the fields of host-parasite co-evolution as well as in sex-associated susceptibility to this infection, and could be an important target for the design of new drugs.

A helminth cestode parasite express an estrogen-binding protein resembling a classic nuclear estrogen receptor.

Helminth β-tubulins are the targets of benzimidazole (BZM) carbamate compounds. The specificity of the interactions between such compounds and their in vivo targets depends on the presence of specific amino acid residues in the target molecules. To discover new and effective anthelmintic drugs, we used a medicinal chemistry approach to synthesize a series of BZM derivatives that exploited the BZM moiety as a template. We have previously found that one compound, 2-(trifluoromethyl)-1H-benzimidazole (RCB20), has better in vitro and in vivo activity than albendazole sulfoxide (ABZSO). In the present study, the effect of RCB20 and ABZSO treatment on expression of Taenia crassiceps cysticerci cytoskeletal proteins such as actin, myosin II, and tubulin isoforms was examined. The effects of RCB20 and ABZSO after 11 days treatment of the parasites was evaluated by light, confocal, and electron microscopy, and by immunochemistry and immunohistochemistry. The RCB20-induced effects were more rapid than the ABZSO-induced effects on the parasites. In the RCB20-treated parasites, we observed gross-structural damage at the whole parasite level, particularly in the inner tissues and flame cells. Changes in the expression patterns of the cytoskeletal proteins, as assessed by immunohistochemistry and immunoblotting, revealed that the most important drug-induced effect on the parasites was a reduction in the expression level of tyrosinated α-tubulins. Our research findings suggest that RCB20 treatment affected posttranslational modification of parasite α-tubulin molecules, which involved removal of the α-tubulin carboxy-terminal tyrosine.

RCB20, an experimental benzimidazole derivative, affects tubulin expression and induces gross anatomical changes in Taenia crassiceps cysticerci.

The Taenia crassiceps ORF strain is used to generate a murine model of cysticercosis, which is used for diagnosis, evaluation of drugs, and vaccination. This particular strain only exists as cysticerci, is easily maintained under in vivo and in vitro conditions, and offers an excellent model for studying the cytoskeletons of cestodes. In this study, several experimental approaches were used to determine the tissue expression of its cytoskeletal proteins. The techniques used were microscopy (video, confocal, and transmission electron), one-dimensional (1D) and two-dimensional (2D) electrophoresis, immunochemistry, and mass spectrometry. The tissue expression of actin, tubulin, and paramyosin was assessed using microscopy, and their protein isoforms were determined with 1D and 2D electrophoresis and immunochemistry. Nineteen spots were excised from a proteomic gel and identified by liquid chromatography-tandem mass spectrometry and immunochemistry. The proteins identified were classic cytoskeletal proteins, metabolic enzymes, and proteins with diverse biological functions, but mainly involved in detoxification activities. Research suggests that most noncytoskeletal proteins interact with actin or tubulin, and the results of the present study suggest that the proteins identified may be involved in supporting the dynamics and plasticity of the cytoskeleton of T. crassiceps cysticerci. These results contribute to our knowledge of the cellular biology and physiology of cestodes.

Analysis of the expression of cytoskeletal proteins of Taenia crassiceps ORF strain cysticerci (Cestoda).

We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment.

Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps.

Trypanosoma cruzi has a particular cytoskeleton that consists of a subpellicular network of microtubules and actin microfilaments. Therefore, it is an excellent target for the development of new anti-parasitic drugs. Benzimidazole 2-carbamates, a class of well-known broad-spectrum anthelmintics, have been shown to inhibit the in vitro growth of many protozoa. Therefore, to find efficient anti-trypanosomal (trypanocidal) drugs, our group has designed and synthesised several benzimidazole derivatives. One, named JVG9 (5-chloro-1H-benzimidazole-2-thiol), has been found to be effective against T. cruzi bloodstream trypomastigotes under both in vitro and in vivo conditions. Here, we present the in vitro effects observed by laser scanning confocal and scanning electron microscopy on T. cruzi trypomastigotes. Changes in the surface and the distribution of the cytoskeletal proteins are consistent with the hypothesis that the trypanocidal activity of JVG9 involves the cytoskeleton as a target.

Article	Year
Actin expression in Taenia solium cysticerci (cestoda): tisular distribution and detection of isoforms. Cell biology international\| PubMed ID: 12972277	2003
Muscular myosin isoforms of Taenia solium (Cestoda). Cell biology international\| PubMed ID: 15566958	2004
In vitro evaluation of the effects of cysticidal drugs in the Taenia crassiceps cysticerci ORF strain using the fluorescent CellTracker CMFDA. Experimental parasitology\| PubMed ID: 20599436	2011
Trypanosoma cruzi: multiple actin isovariants are observed along different developmental stages. Experimental parasitology\| PubMed ID: 20705070	2011
Visualization and 3D reconstruction of flame cells of Taenia solium (cestoda). PloS one\| PubMed ID: 21412407	2011
A helminth cestode parasite express an estrogen-binding protein resembling a classic nuclear estrogen receptor. Steroids\| PubMed ID: 21621550	2011
RCB20, an experimental benzimidazole derivative, affects tubulin expression and induces gross anatomical changes in Taenia crassiceps cysticerci. Parasitology research\| PubMed ID: 23504054	2013
Analysis of the expression of cytoskeletal proteins of Taenia crassiceps ORF strain cysticerci (Cestoda). Parasitology research\| PubMed ID: 24652446	2014
Oestradiol and progesterone differentially alter cytoskeletal protein expression and flame cell morphology in Taenia crassiceps. International journal for parasitology\| PubMed ID: 24879953	2014
JVG9, a benzimidazole derivative, alters the surface and cytoskeleton of Trypanosoma cruzi bloodstream trypomastigotes. Memorias do Instituto Oswaldo Cruz\| PubMed ID: 25230128	2014
JVG9, a benzimidazole derivative, alters the surface and cytoskeleton of Trypanosoma cruzi bloodstream trypomastigotes. Memórias do Instituto Oswaldo Cruz\| PubMed ID: 25317703	2014

Olivia Reynoso-Ducoing

Biography

JoVE Journal Publications

Other Publications