Spy1A is a cyclin-like protein required for progression through the G(1)/S phase of the cell cycle. Elevated Spy1A protein levels have been implicated in tumorigenesis and are attributed to overriding the DNA damage response and enhancing cell proliferation. Understanding how Spy1A is produced and degraded is essential in resolving how it contributes to normal and abnormal growth processes. Herein, we demonstrate that Spy1A is degraded in a cell cycle-dependent manner during mitosis via the ubiquitin-proteasome system. We have resolved the E3 ligase and essential phosphorylation sites mediating Spy1A degradation. Furthermore, we have determined that non-degradable forms of Spy1A do not trigger cell cycle arrest but, rather, contribute to uncontrolled cell growth. Further investigation into the regulation of Spy1A may reveal novel strategies for understanding the etiology and progression of specific growth disorders.
Assisted reproductive technologies (ARTs) are becoming increasingly prevalent and are generally considered to be safe medical procedures. However, evidence indicates that embryo culture may adversely affect the developmental potential and overall health of the embryo. One of the least studied but most important areas in this regard is the effects of embryo culture on epigenetic phenomena, and on genomic imprinting in particular, because assisted reproduction has been linked to development of the human imprinting disorders Angelman and Beckwith-Wiedemann syndromes. In this study, we performed side-by-side comparisons of five commercial embryo culture systems (KSOMaa, Global, Human Tubal Fluid, Preimplantation 1/Multiblast, and G1v5PLUS/G2v5PLUS) in relation to a best-case (in vivo-derived embryos) and a worst-case (Whitten culture) scenario. Imprinted DNA methylation and expression were examined at three well-studied loci, H19, Peg3, and Snrpn, in mouse embryos cultured from the 2-cell to the blastocyst stage. We show that embryo culture in all commercial media systems resulted in imprinted methylation loss compared to in vivo-derived embryos, although some media systems were able to maintain imprinted methylation levels more similar to those of in vivo-derived embryos in comparison to embryos cultured in Whitten medium. However, all media systems exhibited loss of imprinted H19 expression comparable to that using Whitten medium. Combined treatment of superovulation and embryo culture resulted in increased perturbation of genomic imprinting, above that from culture alone, indicating that multiple ART procedures further disrupt genomic imprinting. These results suggest that time in culture and number of ART procedures should be minimized to ensure fidelity of genomic imprinting during preimplantation development.
Assisted reproductive technologies are fertility treatments used by subfertile couples to conceive their biological child. Although generally considered safe, these pregnancies have been linked to genomic imprinting disorders, including Beckwith–Wiedemann and Silver–Russell Syndromes. Silver–Russell Syndrome is a growth disorder characterized by pre- and post-natal growth retardation. The Mest imprinted domain is one candidate region on chromosome 7 implicated in Silver–Russell Syndrome. We have previously shown that maintenance of imprinted methylation was disrupted by superovulation or embryo culture during pre-implantation mouse development. For superovulation, this disruption did not originate in oogenesis as a methylation acquisition defect. However, in comparison to other genes, Mest exhibits late methylation acquisition kinetics, possibly making Mest more vulnerable to perturbation by environmental insult. In this study, we present a comprehensive evaluation of the effects of superovulation and in vitro culture on genomic imprinting at the Mest gene. Superovulation resulted in disruption of imprinted methylation at the maternal Mest allele in blastocysts with an equal frequency of embryos having methylation errors following low or high hormone treatment. This disruption was not due to a failure of imprinted methylation acquisition at Mest in oocytes. For cultured embryos, both the Fast and Slow culture groups experienced a significant loss of maternal Mest methylation compared to in vivo-derived controls. This loss of methylation was independent of development rates in culture. These results indicate that Mest is more susceptible to imprinted methylation maintenance errors compared to other imprinted genes.
Genomic imprinting is a specialized transcriptional mechanism that results in the unequal expression of alleles based on their parent-of-origin. Many imprinted genes have been shown to be critical for proper embryonic and fetal development, and disruption of genomic imprinting at these loci has been associated with many development disorders. Recently, increased frequencies of many imprinting disorders have been correlated with the use of assisted reproductive technologies (ARTs). Rigorous and thorough testing of ARTs is required to determine their influence on processes of genomic imprinting. I hypothesize that the acquisition of genomic imprinting in developing germ cells is disrupted by superovulation, and that imprinting maintenance mechanisms are disrupted during early mouse development by culture media used in human ARTs. We have developed a method to evaluate the methylation and expression patterns of known imprinted genes from individual blastocysts. We utilized this technique to separately address the effects of two different hormone dosages, and six different culture media on imprinted methylation and expression. Superovulation resulted in a loss of imprinted methylation at both hormone concentrations tested, in a dose-dependent manner, and embryo culture resulted in a loss of imprinted methylation in all media tested. We conclude that both imprint acquisition and imprint maintenance can be perturbed by assisted reproductive technologies. Further analysis and use of our novel technique will allow more accurate and comprehensive study of the epigenetic effects of various environmental insults on the preimplantation embryo. (poster)
The present chapter proposes that understanding mammalian affective and defensive responses can significantly contribute to an understanding of dissociative phenomena including the traumatic separation of self-states. We believe this is crucial to understanding peritraumatic responses, posttraumatic stress disorder (PTSD), dissociative disorders, somatization and somatoform (e.g., conversion) disorders, and many disorders of emotion regulation. We argue that the neuroscientific understanding of dissociative disorders is best served clinically by a focus on the brain areas controlling the generation of basic affects and defense responses rather than on the output changes in the autonomic nervous system. The brainstem focus includes not only areas for rapid response to threat but also those necessary for seeking and making the interpersonal connections preceding attachment. This offers hypotheses on the nervous system substrates of disorganized attachments and their clinical consequences (e.g., dissociative disorders).
Currently, the stage of embryo development has been proposed as one of many criteria for identifying healthy embryos in infertility clinics with the fastest embryos being highlighted as the healthiest. However the validity of this as an accurate criterion with respect to genomic imprinting is unknown. Given that embryo development in culture generally requires an extra day compared to in vivo development, we hypothesized that loss of imprinting correlates with slower rates of embryonic development. To evaluate this, embryos were recovered at the 2-cell stage, separated into four groups based on morphological stage at two predetermined time points, and cultured to blastocysts. We examined cell number, embryo volume, embryo sex, imprinted Snrpn and H19 methylation, imprinted Snrpn, H19, and Cdkn1c expression, and expression of genes involved in embryo metabolism-Atp1a1, Slc2a1, and Mapk14-all within the same individual embryo. Contrary to our hypothesis, we observed that faster developing embryos exhibited greater cell numbers and embryo volumes as well as greater perturbations in genomic imprinting and metabolic marker expression. Embryos with slower rates of preimplantation development were most similar to in vivo derived embryos, displaying similar cell numbers, embryo volumes, Snrpn and H19 imprinted methylation, H19 imprinted expression, and Atp1a1 and Slc2a1 expression. We conclude that faster development rates in vitro are correlated with loss of genomic imprinting and aberrant metabolic marker expression. Importantly, we identified a subset of in vitro cultured embryos that, according to the parameters evaluated, are very similar to in vivo derived embryos and thus are likely most suitable for embryo transfer.
Primary adenocarcinoma of the oropharynx most often arises from the minor salivary glands, and primary squamous cell carcinoma is more commonly seen arising from the tongue. Few cases of adenocarcinoma not otherwise specified of the tongue have been reported in the literature, and none found on the dorsum of the tongue. Successful treatment strategies have therefore not been defined.We report a case of adenocarcinoma located on the dorsum of the posterior one third of the tongue adjacent to the circumvallate papillae in a woman presenting with globus sensation and mild dysphagia. Treatment consisted of transoral laser excision and postoperative external-beam radiotherapy, resulting in disease-free survival at her 5-year follow-up. The goals of this report are to present a case of adenocarcinoma arising from the minor salivary gland located on the dorsum of the tongue, to discuss previous reports of similar cases, and to suggest that surgery with or without radiotherapy be used as the mainstay of treatment.
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