924 resultados para Oocyte competence


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Tumors that metastasize do so to preferred target organs. To explain this apparent specificity, Paget, > 100 years ago, formulated his seed and soil hypothesis; i.e., the cells from a given tumor would "seed'' only favorable "soil'' offered by certain groups. The hypothesis implies that cancer cells must find a suitable "soil'' in a target organ--i.e., one that supports colonization--for metastasis to occur. We demonstrate in this report that ability of human colon cancer cells to colonize liver tissue governs whether a particular colon cancer is metastatic. In the model used in this study, human colon tumors are transplanted into the nude mouse colon as intact tissue blocks by surgical orthotopic implantation. These implanted tumors closely simulate the metastatic behavior of the original human patient tumor and are clearly metastatic or nonmetastatic to the liver. Both classes of tumors were equally invasive locally into tissues and blood vessels. However, the cells from each class of tumor behave very differently when directly injected into nude mouse livers. Only cells from metastasizing tumors are competent to colonize after direct intrahepatic injection. Also, tissue blocks from metastatic tumors af fixed directly to the liver resulted in colonization, whereas no colonization resulted from nonmetastatic tumor tissue blocks even though some growth occurred within the tissue block itself. Thus, local invasion (injection) and even adhesion to the metastatic target organ (blocks) are not sufficient for metastasis. The results suggest that the ability to colonize the liver is the governing step in the metastasis of human colon cancer.

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The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P-antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies.

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Competence for genetic transformation in Streptococcus pneumoniae has been known for three decades to arise in growing cultures at a critical cell density, in response to a secreted protease-sensitive signal. We show that strain CP1200 produces a 17-residue peptide that induces cells of the species to develop competence. The sequence of the peptide was found to be H-Glu-Met-Arg-Leu-Ser-Lys-Phe-Phe-Arg-Asp-Phe-Ile-Leu-Gln-Arg- Lys-Lys-OH. A synthetic peptide of the same sequence was shown to be biologically active in small quantities and to extend the range of conditions suitable for development of competence. Cognate codons in the pneumococcal chromosome indicate that the peptide is made ribosomally. As the gene encodes a prepeptide containing the Gly-Gly consensus processing site found in peptide bacteriocins, the peptide is likely to be exported by a specialized ATP-binding cassette transport protein as is characteristic of these bacteriocins. The hypothesis is presented that this transport protein is encoded by comA, previously shown to be required for elaboration of the pneumococcal competence activator.

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Xenopus oocytes are a valuable aid for studying the molecular structure and function of ionic channels and neurotransmitter receptors. Their use has recently been extended by the demonstration that oocytes can incorporate foreign membranes carrying preassembled receptors and channels. Here we show that when reconstituted in an artificial lipid matrix and injected into Xenopus oocytes, purified nicotinic acetylcholine receptors are efficiently inserted into the plasma membrane, where they form "clusters" of receptors that retain their native properties. This constitutes an innovative approach that, besides allowing the analyses of membrane fusion processes, is also a powerful technique for studying the characteristics and regulation of many membrane proteins (with their native stoichiometry and configuration) upon reinsertion into the membrane of a very convenient host cell system.

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The Gram-negative bacterial pathogen Neisseria gonorrhoeae is naturally competent for transformation with species-related DNA. We show here that two phase-variable pilus-associated proteins, the major pilus subunit (pilin, or PilE) and PilC, a factor known to function in the assembly and adherence of gonococcal pili, are essential for transformation competence. The PilE and PilC proteins are necessary for the conversion of linearized plasmid DNA carrying the Neisseria-specific DNA uptake signal into a DNase-resistant form. The biogenesis of typical pilus fibers is neither essential nor sufficient for this process. DNA uptake deficiency of defined piliated pilC1,2 double mutants can be complemented by expression of a cloned pilC2 gene in trans. The PilC defect can also be restored by the addition of purified PilC protein, or better, pili containing PilC protein, to the mutant gonococci. Our data suggest that the two phase-variable Pil proteins act on the bacterial cell surface and cooperate in DNA recognition and/or outer membrane translocation.

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The enzyme collagenase (EC 3.4.24.7), a key mediator in biological remodeling, can be induced in early-passage fibroblasts by a wide variety of agents and conditions. In contrast, at least some primary tissue fibroblasts are incompetent to synthesize collagenase in response to many of these stimulators. In this study, we investigate mechanisms controlling response to two of the conditions in question: (i) trypsin or cytochalasin B, which disrupt actin stress fibers, or (ii) phorbol 12-myristate 13-acetate (PMA), which activates growth factor signaling pathways. We demonstrate that collagenase expression stimulated by trypsin or cytochalasin B is regulated entirely through an autocrine cytokine, interleukin 1 alpha (IL-1 alpha). The IL-1 alpha intermediate also constitutes the major mechanism by which PMA stimulates collagenase expression, although a second signaling pathway(s) contributes to a minor extent. Elevation of the IL-1 alpha level in response to stimulators is found to be sustained by means of an autocrine feedback loop in early-passage fibroblast cultures. In contrast, fibroblasts freshly isolated from the tissue are incompetent to activate and sustain the IL-1 alpha feedback loop, even though they synthesize collagenase in response to exogenous IL-1. We conclude that this is the reason why tissue fibroblasts are limited, in comparison with subcultured fibroblasts, in their capacity to synthesize collagenase. Activation of the IL-1 alpha feedback loop, therefore, seems likely to be an important mechanism by which resident tissue cells adopt the remodeling phenotype.

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We have developed a paracrine signaling assay capable of mimicking inductive events in the early vertebrate embryo. RNA encoding one or more secreted proteins is microinjected into a Xenopus laevis oocyte. After a brief incubation to allow translation, a piece of embryonic tissue competent to respond to the signaling protein is grafted onto the oocyte. The secreted protein's effect on the grafted explant is then scored by assaying expression of tissue-specific markers. Explants of ectodermal tissue from blastula or gastrula stage embryos were grafted onto oocytes that had been injected with RNA encoding activin or noggin. We found that the paracrine assay faithfully reconstitutes mesoderm induction by activin and neural induction by noggin. Blastula-stage explants grafted onto activin-expressing oocytes expressed the mesodermal marker genes brachyury, goosecoid, and muscle actin. Gastrula-stage explants grafted onto noggin-expressing oocytes expressed neural cell adhesion molecule (NCAM) and formed cement gland. By injecting pools of RNA synthesized from a cDNA expression library into the oocyte, we also used the assay to screen for secreted neural-inducing proteins. We assayed 20,000 independent transformants of a library constructed from LiCl-dorsalized Xenopus laevis embryos, and we identified two cDNAs that induced neural tissue in ectodermal explants from gastrula-stage embryos. Both cDNAs encode noggin. These results suggest that the paracrine assay will be useful for the cloning of novel signaling proteins as well as for the analysis of known factors.

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We have studied the functional consequences of a mutation in the epithelial Na+ channel that causes a heritable form of salt-sensitive hypertension, Liddle disease. This mutation, identified in the original kindred described by Liddle, introduces a premature stop codon in the channel beta subunit, resulting in a deletion of almost all of the C terminus of the encoded protein. Coexpression of the mutant beta subunit with wild-type alpha and gamma subunits in Xenopus laevis oocytes resulted in an approximately 3-fold increase in the macroscopic amiloride-sensitive Na+ current (INa) compared with the wild-type channel. This change in INa reflected an increase in the overall channel activity characterized by a higher number of active channels in membrane patches. The truncation mutation in the beta subunit of epithelial Na+ channel did not alter the biophysical and pharmacological properties of the channel--including unitary conductance, ion selectivity, or sensitivity to amiloride block. These results provide direct physiological evidence that Liddle disease is related to constitutive channel hyperactivity in the cell membrane. Deletions of the C-terminal end of the beta and gamma subunits of rat epithelial Na+ channel were functionally equivalent in increasing INa, suggesting that the cytoplasmic domain of the gamma subunit might be another molecular target for mutations responsible for salt-sensitive forms of hypertension.

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The aim of a university education is the intellectual development of citizens, and the training of professionals for their subsequent entry into the workforce. However, this entry into the workforce, following the theoretical education usually provided by the university, implies that students have to manage this difficult transition by themselves. Society, in a continual process of transformation, requires of universities that they adjust, adapting the education they offer to comply with the demands of society and the workplace. Socio-emotional skills would seem to have influence predicting professional performance. These skills also influence job-finding and employability. Consequently, providing teachers with an education in socioemotional competences is becoming a necessary task within universities, and the majority of teaching staff consider these skills to be fundamental to the personal and socio-emotional development of students. The objective of our proposed work is to establish the characteristic profile of competences of a sample of teachers in training, and compare it with the competences profile of graduate students belonging to the fields of law sciences, social sciences, humanities, science and technology, and health. Starting from results, implications will be derived for the development of generic competences of socio-emotional type in the framework of the European Frame of Higher Education.

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Objective. To determine the level of involvement of clinical nurses accredited by the Universitat Jaume I (Spain) as mentors of practice (Reference Nurses) in the evaluation of competence of nursing students. Methodolgy. Cross-sectional study, in which the “Clinical Practice Assessment Manual” (CPAM) reported by reference 41 nurses (n=55) were analyzed. Four quality criteria for completion were established: with information at least 80% of the required data, the presence of the signature and final grade in the right place. Verification of learning activities was also conducted. Data collection was performed concurrently reference for nurses and teachers of the subjects in the formative evaluations of clinical clerkship period in the matter “Nursing Care in Healthcare Processes “, from March to June 2013. Results. 63% of CPAM were completed correctly, without reaching the quality threshold established (80%). The absence of the signature is the main criteria of incorrect completion (21%). Nine learning activities do not meet the quality threshold set (80%) (p < 0.05). There are significant differences according to clinical units p < 0.05. From the 30 learning activities evaluated in the CPAM, it can be stated that nine of them do not reach the verification threshold established (80%), therefore it cannot be assumed that these activities had been completed by students and evaluated by the RefN throughout the clinical clerkship period. Conclusion. The level of involvement of Reference Nurse cannot be considered adequate, although strategies to encourage involvement through collaboration and training must be developed.

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In this editorial the author describes the essential nature and characteristics of cultural competence.

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Three-page handwritten essay in English by Buckminster with a story about a wealthy young Englishman named Francis who discovers that money is not the source of happiness. The essay is titled with a quote from Edward Young's poem, "The Complaint." Buckminster's essay begins, "Francis was the son of a rich English nobleman."

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PAWP, a candidate sperm-borne oocyte activating factor, induces oocyte activation and acts upstream of the calcium signalling pathway, however, PAWP’s downstream signalling pathway in oocyte cytoplasm remains to be uncovered. Data from our lab suggested that the interacting partner of PAWP, at least in the frog (Xenopus laevis) model may be YAP, a highly expressed protein in amphibian and mammalian oocytes. Therefore, the objectives of this study were to confirm that PAWP’s predominant binding partner in Xenopus laevis oocyte is YAP; to determine if mammalian oocyte activation is also dependent on PAWP-YAP interaction; and to verify that the PAWP-YAP interaction during oocyte activation is dependent on the WWI domain module. By immunohistochemistry, YAP was localized predominantly in the cytosol of metaphase II-arrested Xenopus laevis oocytes, where presumably the PAWP-YAP interaction occurs. Utilizing Far Western blotting, YAP was identified as the predominant binding partner of PAWP, in metaphase II-arrested frog (Xenopus laevis), swine (Sus scrofa) and mouse (mus musculus) oocytes. The specificity of this interaction was then tested on Far Western blotting of mouse ovarian and oocyte cytosolic extracts, by competition with both wild-type and point-mutated recombinant WWI domains derived from YAP. The removal of GST from the wild-type WWI-GST fusion protein was a requirement for effective blockage of WWI module interaction between PAWP and YAP. As expected, the mutated WWI domain was ineffective in inhibiting the PAWP-YAP interaction. To conclude, this study identified YAP as the predominant binding partner of PAWP in both amphibian and mammalian oocytes, and showed this interaction is dependent on the WWI modular interaction. The results allow us to test the functional relevance of this WWI modular interaction during oocyte activation in vivo, in the future.

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The purpose of the current dissertation is to identify the features of effective interventions by exploring the experiences of youth with ASD who participate in such interventions, through two intervention studies (Studies 1 and 2) and one interview study (Study 3). Studies 1 and 2 were designed to support the development of social competence of youth with ASD through Structured Play with LEGO TM (Study 1, 12 youths with ASD, ages 7–12) and Minecraft TM (Study 2, 4 youths with ASD, ages 11–13). Over the course of the sessions, the play of the youth developed from parallel play (children playing alone, without interacting) to co-operative play (playing together with shared objectives). The results of Study 2 showed that rates of initiations and levels of engagement increased from the first session to the final session. In Study 3, 12 youths with ASD (ages 10–14) and at least one of their parents were interviewed to explore what children and their parents want from programs designed to improve social competence, which activities and practices were perceived to promote social competence by the participants, and which factors affected their decisions regarding these programs. The adolescents and parents looked for programs that supported social development and emotional wellbeing, but did not always have access to the programs they would have preferred, with factors such as cost and location reducing their options. Three overarching themes emerged through analysis of the three studies: (a) interests of the youth; (b) structure, both through interactions and instruction; and (c) naturalistic settings. Adolescents generally engage more willingly in interventions that incorporate their interests, such as play with Minecraft TM in Study 2. Additionally, Structured Play and structured instruction were crucial components of providing safe and supportive contexts for the development of social competence. Finally, skills learned in naturalistic settings tend to be applied more successfully in everyday situations. The themes are analysed through the lens of Vygotsky’s (1978) perspectives on learning, play, and development. Implications of the results for practitioners and researchers are discussed.