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OBJECTIVE: To analyze dizygotic twinning rates and outcomes over a 25-year period. METHODS: Birth and fetal death certificates from 1980-2004 in Washington State, USA, were analyzed retrospectively to find factors associated with the increase in sex-discordant twins through time. 'Low' and 'high' fertility treatment groups were defined according to demographic traits. Perinatal mortality was defined as fetal or neonatal death of one or both twins and Weinberg's rule was used to estimate mortality for monozygotic and dizygotic pairs. RESULTS: Controlling simultaneously for maternal age, race, parity, and education did not eliminate the trend of increasing sex-discordant twins from 1992-2004 (M-H chi2 P=0.001). The 'low' fertility group had a non-significant decline in sex-discordant twins (M-H chi2 P=0.24), whereas the 'high' fertility group had a significant increase (M-H chi2 P=0.001). Perinatal mortality decreased for monozygtic twin pairs throughout the study period, but decreased until the mid-1990s and then increased slightly through 2004 for the dizygotic twin pairs. CONCLUSION: Advancing maternal age and increasing use of fertility treatments are largely responsible for the increase in dizygotic twins from 1980-2004 and may also be responsible for the stalling of the decline in perinatal mortality rate. [ABSTRACT FROM AUTHOR]

Background: The University of New Mexico School of Medicine established the combined baccalaureate/medical degree (BA/MD) program in response to critical physician shortages in New Mexico (NM). This 8-year program aims to improve health care in NM by expanding access to medical education for local students, particularly from rural and underserved communities and/or racial/ethnically underrepresented in medicine (URiM) in NM., Objectives: To describe the BA/MD program's initial design, the impact of improvements on retention, and the outcomes in terms of physicians in practice, particularly in primary care specialties., Methods: The study reviews the BA/MD program's progress from 2006 to 2023, focusing on curriculum and support enhancements. Retention rates and choice of primary care specialties were analyzed by geographic origin and racial/ethnic background., Results: From 2006 to 2023, the program graduated 81 physicians, with 53 practicing in 10 of NM's 33 counties. Approximately two-thirds specialize in primary care, and a similar proportion are URiM. Students from 31 of NM's 33 counties were admitted, with two-thirds coming from outside the state's metropolitan area. Overall retention and retention across demographic groups improved significantly in the baccalaureate phase of the program due to changes in curriculum and support services., Conclusions: The program has effectively addressed physician shortages in NM, particularly in rural and underserved areas. Its success in training and retaining physicians from diverse backgrounds, with a focus on primary care, is crucial for improving health care access in the state. Ongoing improvements in the program are essential to sustaining and enhancing these outcomes., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Many medical schools in the United States have integrated anatomy into an organ-based preclinical curriculum with some schools using anatomy as the cornerstone of their reorganization efforts. Curricular change could affect one or more of the three domains of learning, with the cognitive domain often scrutinized exclusively. A previous study reported the impact of anatomy integration on the affective domain, specifically, student attitudes toward learning anatomy. This mixed methods follow-up study asked if the observed attitudinal changes and lack of effect on student knowledge and confidence persisted using knowledge and confidence surveys, focus groups, internal and national surveys, and United States Medical Licensing Examination ® performance metrics. Results evidenced the persistence of specific attitudinal differences between cohorts with blocked versus integrated anatomy with no apparent short- or long-term differences in anatomy learning or confidence in this learning. Altered attitudes included lower value placed on working in teams and reflective practices, and less recognition of anatomy's contribution (or less contribution of anatomy learning) to professional identity formation. These attitudinal changes could result in a weaker foundation for building collaborative skills throughout the medical curriculum. A decreased sense of student engagement also followed curricular change, as assessed by data from the American Association of Medical Colleges Year 2 Questionnaire. Overall, results emphasized the necessity of anticipating, monitoring, and if necessary, addressing changes in the affective domain when undertaking curricular change., (© 2024 American Association for Anatomy.)

RNA binding proteins (RBPs) post-transcriptionally regulate gene expression by associating with regulatory sequences in the untranslated regions of mRNAs. Cold-inducible RBP (CIRP) is a stress-induced RBP that was recently shown to modulate inflammation in response to cellular stress, where it increases or decreases pro-tumorigenic (proinflammatory) cytokines in different contexts. CIRP expression is altered in several cancers, including breast cancer, but the effects of CIRP on inflammation in breast cancer is not known. Here, we investigate if CIRP alters growth and the inflammatory profile of breast tumors. Transgenic mice overexpressing CIRP in the mammary epithelium were crossed with the PyMT mouse model of breast cancer, and the effects on both early and late tumorigenesis and inflammation were assessed. The effects of CIRP knockdown were also assessed in Py2T cell grafts. Overexpression of CIRP led to decreased tumorigenesis in the PyMT mouse model. Conversely, the knockdown of CIRP in Py2T cell grafts led to increased tumor growth. Luminex cytokine assays assessed the effects on the inflammatory environment. CIRP/PyMT mammary glands/mammary tumors and serum had decreased cytokines that promote inflammation, angiogenesis, and metastasis compared to PyMT mammary glands and serum, documenting a shift towards an environment less supportive of tumorigenesis. CIRP overexpression also decreased CD4 + helper T cells and increased CD8 + cytotoxic T cells in mammary tumors. Overall, these data support a role for CIRP as a potent antitumor molecule that suppresses both local and systemic pro-tumorigenic inflammation.

Context: During pregnancy, the immune system is altered, putting women at increased risk of complications from respiratory viruses. There is limited information about the effects of COVID-19 on pregnant women and obstetrics programs, particularly in rural regions., Aims: Most published reports have been from large urban hospitals. This study sought to gain insight into how the COVID-19 pandemic is impacting rural obstetrics programs., Materials and Methods: This qualitative study surveyed participants who worked in rural obstetrics programs in New Mexico using a free response questionnaire., Results: The pandemic has changed the obstetrics experience in rural New Mexico by impacting the relationship between patients and providers and altering the family-centered model of birth. Difficulties unique to rural obstetrics included lack of transportation, limited hospital rooms and limited staff, including OB providers, nurses and housekeeping. Wellness has been impacted for providers, manifested in increased anxiety, stress and burnout. Patients reported increased anxiety, decreased family support, and potential disruptions in maternal-infant bonding., Conclusions: Rural hospitals and clinics in New Mexico and across the United States operate with challenges at baseline, with many clinics across the nation closing, leading to increasing shortages of healthcare services in rural areas. This study showed that COVID-19 has increased the challenges that rural obstetrics providers face, altering the way they practice and creating uncertainty for the future due to potentially permanent changes to their practice. Future research will explore the lasting effects of the pandemic on rural obstetrics providers specifically, and rural hospitals generally., Competing Interests: There are no conflicts of interest., (Copyright: © 2021 Journal of Family Medicine and Primary Care.)

Anatomy is fundamental to clinical practice, is considered a rite of passage in becoming a physician and is key to professional identity formation. The anatomy course that began the medical curriculum at the University of New Mexico School of Medicine was recently dissolved to address content and process gaps in the pre-clinical curriculum. Anatomy was integrated into the organ system blocks to make room for new courses to address content gaps. Previous reports of anatomy integration document more positive attitudes and perceptions to teaching anatomy in context, as compared to an independent course. The current prospective study compared two medical student cohorts to determine the effects of teaching anatomy in and out of context on the cognitive and affective domains of learning. In a pre, post, and follow-up design, methods included content assessments, confidence probes, and attitude surveys informed by focus groups. Results indicated that anatomical knowledge and student confidence was gained and mastered in both curricula. Initial acquisition of content was higher in the integrated curriculum, but not maintained. Students in the integrated curriculum displayed a different relationship to learning anatomy, appearing more concerned with their personal progression than with the connection of anatomy to medical practice or patient care. These students also agreed less with statements related to working in teams, reflective practices and professional identity formation. Further studies will determine if this difference will diminish with continued exposure to anatomy and may inform future curricular adjustments. Anat Sci Educ. © 2018 American Association of Anatomists., (© 2018 American Association of Anatomists.)

RNA binding proteins (RBPs) play key roles in RNA dynamics, including subcellular localization, translational efficiency and metabolism. Cold-inducible RNA binding protein (CIRP) is a stress-induced protein that was initially described as a DNA damage-induced transcript (A18 hnRNP), as well as a cold-shock domain containing cold-stress response protein (CIRBP) that alters the translational efficiency of its target messenger RNAs (mRNAs). This review summarizes recent work on the roles of CIRP in the context of inflammation and cancer. The function of CIRP in cancer appeared to be solely driven though its functions as an RBP that targeted cancer-associated mRNAs, but it is increasingly clear that CIRP also modulates inflammation. Several recent studies highlight roles for CIRP in immune responses, ranging from sepsis to wound healing and tumor-promoting inflammation. While modulating inflammation is an established role for RBPs that target cytokine mRNAs, CIRP appears to modulate inflammation by several different mechanisms. CIRP has been found in serum, where it binds the TLR4-MD2 complex, acting as a Damage-associated molecular pattern (DAMP). CIRP activates the NF-κB pathway, increasing phosphorylation of Iκκ and IκBα, and stabilizes mRNAs encoding pro-inflammatory cytokines. While CIRP promotes higher levels of pro-inflammatory cytokines in certain cancers, it also decreases inflammation to accelerate wound healing. This dichotomy suggests that the influence of CIRP on inflammation is context dependent and highlights the importance of detailing the mechanisms by which CIRP modulates inflammation. WIREs RNA 2018, 9:e1462. doi: 10.1002/wrna.1462 This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications., (© 2018 Wiley Periodicals, Inc.)

Objective: To investigate maternal and infant outcomes associated with delivery mode for twins with a cephalic presenting twin., Methods: Linked birth certificate and ICD hospital discharge data were analyzed retrospectively for 5573 mothers and their respective twin pairs born live at 34-42 weeks' gestation, with twin A vertex, in Washington State from 1997-2007. Relative risks (RR) and 95% confidence intervals of adverse maternal and twin pair outcomes were calculated for vaginal delivery or cesarean during labor in comparison to cesarean without labor., Results: Vaginal delivery or cesarean during labor was associated with significantly elevated rates of maternal hemorrhage (RR = 2.8 [2.2,3.7]), infection (RR = 2.2 [1.5,3.3]), twin pair birth injury (RR = 2.6 [1.2,5.4]) and low 5-min Apgar scores (RR = 1.4 [1.1,1.8]) and with significantly lower rates of ventilation among preterm twin pairs only (RR = 0.8 [0.7,0.9]). The lowest rate of combined poor short-term outcomes occurred in mothers and twin pairs delivered by cesarean without labor (23%) and the highest rates occurred in those with operative vaginal or cesarean during labor (39% and 34%, respectively). Among women in labor, 35% of nulliparas achieved spontaneous vaginal delivery of both twins compared to 63% of non-nulliparas., Conclusion: For nulliparous women who carry twins to term, planned cesarean may improve outcomes.

RNA binding proteins (RBPs) regulate gene expression by controlling mRNA export, translation, and stability. When altered, some RBPs allow cancer cells to grow, survive, and metastasize. Cold-inducible RNA binding protein (CIRP) is overexpressed in a subset of breast cancers, induces proliferation in breast cancer cell lines, and inhibits apoptosis. Although studies have begun to examine the role of CIRP in breast and other cancers, its role in normal breast development has not been assessed. We generated a transgenic mouse model overexpressing human CIRP in the mammary epithelium to ask if it plays a role in mammary gland development. Effects of CIRP overexpression on mammary gland morphology, cell proliferation, and apoptosis were studied from puberty through pregnancy, lactation and weaning. There were no gross effects on mammary gland morphology as shown by whole mounts. Immunohistochemistry for the proliferation marker Ki67 showed decreased proliferation during the lactational switch (the transition from pregnancy to lactation) in mammary glands from CIRP transgenic mice. Two markers of apoptosis showed that the transgene did not affect apoptosis during mammary gland involution. These results suggest a potential in vivo function in suppressing proliferation during a specific developmental transition., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Luminescent semiconductor nanocrystals, also known as quantum dots (QD), possess highly desirable optical properties that account for development of a variety of exciting biomedical techniques. These properties include long-term stability, brightness, narrow emission spectra, size tunable properties and resistance to photobleaching. QD have many promising applications in biology and the list is constantly growing. These applications include DNA or protein tagging for in vitro assays, deep-tissue imaging, fluorescence resonance energy transfer (FRET), and studying dynamics of cell surface receptors, among others. Here we explored the potential of QD-mediated labeling for the purpose of tracking an intracellular protein inside live cells., Results: We manufactured dihydrolipoic acid (DHLA)-capped CdSe-ZnS core-shell QD, not available commercially, and coupled them to the cell cycle regulatory protein Cyclin E. We then utilized the QD fluorescence capabilities for visualization of Cyclin E trafficking within cells of Xenopus laevis embryos in real time., Conclusions: These studies provide "proof-of-concept" for this approach by tracking QD-tagged Cyclin E within cells of developing embryos, before and during an important developmental period, the midblastula transition. Importantly, we show that the attachment of QD to Cyclin E did not disrupt its proper intracellular distribution prior to and during the midblastula transition. The fate of the QD after cyclin E degradation following the midblastula transition remains unknown.

RNA binding protein (RBPs) and microRNAs (miRNAs or miRs) are post-transcriptional regulators of gene expression that are implicated in development of cancers. Although their individual roles have been studied, the crosstalk between RBPs and miRNAs is under intense investigation. Here, we show that in breast cancer cells, cyclin E1 upregulation by the RBP HuR is through specific binding to regions in the cyclin E1 mRNA 3' untranslated region (3'UTR) containing U-rich elements. Similarly, miR-16 represses cyclin E1, dependent on its cognate binding sites in the cyclin E1 3'UTR. Evidence in the literature indicates that HuR can regulate miRNA expression and recruit or dissociate RNA-induced silencing complexes (RISC). Despite this, miR-16 and HuR do not affect the other's expression level or binding to the cyclin E1 3'UTR. While HuR overexpression partially blocks miR-16 repression of a reporter mRNA containing the cyclin E1 3'UTR, it does not block miR-16 repression of endogenous cyclin E1 mRNA. In contrast, miR-16 blocks HuR-mediated upregulation of cyclin E1. Overall our results suggest that miR-16 can override HuR upregulation of cyclin E1 without affecting HuR expression or association with the cyclin E1 mRNA.

The mammary gland is one of the best-studied examples of an organ whose structure and function are influenced by reciprocal signaling and communication between cells and their microenvironment. The mammary epithelial cell (MEC) microenvironment includes stromal cells and extracellular matrix (ECM). Abundant evidence shows that the ECM and growth factors co-operate to regulate cell cycle progression, and that the ECM is altered in breast tumors. In particular, mammographically dense breast tissue is a significant risk factor for developing breast carcinomas. Dense breast tissue is associated with increased stromal collagen and epithelial cell content. In this article, we overview recent studies addressing the effects of ECM composition on the breast cancer cell cycle. Although the normal breast ECM keeps the MEC cycle in check, the ECM remodeling associated with breast cancer positively regulates the MEC cycle. ECM effects on the downstream biochemical and mechanosignaling pathways in both normal and tumorigenic MECs will be reviewed., (Copyright © 2012 Wiley Periodicals, Inc.)

Cyclins are regulatory subunits that bind to and activate catalytic Cdks. Cyclin E associates with Cdk2 to mediate the G1/S transition of the cell cycle. Cyclin E is overexpressed in breast, lung, skin, gastrointestinal, cervical, and ovarian cancers. Its overexpression correlates with poor patient prognosis and is involved in the etiology of breast cancer. We have been studying how cyclin E is normally downregulated during development in order to determine if disruption of similar mechanisms could either contribute to its overexpression in cancer, or be exploited to decrease its expression. In Xenopus laevis embryos, cyclin E protein level is high and constant until its abrupt destabilization by an undefined mechanism after the 12th cell cycle, which corresponds to the midblastula transition (MBT) and remodeling of the embryonic to the adult cell cycle. Since degradation of mammalian cyclin E is regulated by the ubiquitin proteasome system and is phosphorylation dependent, we examined the role of phosphorylation in Xenopus cyclin E turnover. We show that similarly to human cyclin E, phosphorylation of serine 398 and threonine 394 plays a role in cyclin E turnover at the MBT. Immunofluorescence analysis shows that cyclin E relocalizes from the cytoplasm to the nucleus preceding its degradation. When nuclear import is inhibited, cyclin E stability is markedly increased after the MBT. To investigate whether degradation of Xenopus cyclin E is mediated by the proteasomal pathway, we used proteasome inhibitors and observed a progressive accumulation of cyclin E in the cytoplasm after the MBT. Ubiquitination of cyclin E precedes its proteasomal degradation at the MBT. These results show that cyclin E destruction at the MBT requires both phosphorylation and nuclear import, as well as proteasomal activity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The behavior of breast epithelial cells is influenced by their microenvironment which includes stromal cells and extracellular matrix (ECM). During cancer progression, the tissue microenvironment fails to control proliferation and differentiation, resulting in uncontrolled growth and invasion. Upon invasion, the ECM encountered by breast cancer cells changes from primarily laminin and collagen IV to primarily collagen I. We show here that culturing invasive breast cancer cells in 3-dimensional (3D) collagen I inhibits proliferation through direct regulation of cyclin E1, a G(1)/S regulator that is overexpressed in breast cancer. When the breast cancer cell line MDA-MB-231 was cultured within 3D collagen I gels, the G(1)/S transition was inhibited as compared to cells cultured on conventional 2D collagen or plastic dishes. Cells in 3D collagen downregulated cyclin E1 protein and mRNA, with no change in cyclin D1 level. Cyclin D1 was primarily cytoplasmic in 3D cultures, and this was accompanied by decreased phosphorylation of Rb, a nuclear target for both cyclin E1- and cyclin D1-associated kinases. Positive regulators of cyclin E1 expression, the transcription factor c-Myc and cold-inducible RNA binding protein (CIRP), were decreased in 3D collagen cultures, while the collagen I receptor β1 integrin was greatly increased. Inhibition of β1 integrin function rescued proliferation and cyclin E1 expression as well as c-Myc expression and Rb phosphorylation, but cyclin D1 remained cytoplasmic. We conclude that cyclin E1 is repressed independent of effects on cyclin D1 in a 3D collagen environment and dependent on β1 integrin interaction with collagen I, reducing proliferation of invasive breast cancer cells.

Objective: To examine trends in twin gestational age over time, with adjustment for potential confounding factors, and to assess twin pair mortality and respiratory support over time., Methods: Rates of preterm births, respiratory support, and neonatal mortality were calculated for 21,569 twin pairs born from 1980 to 2005 in Washington State, using birth certificate and hospital discharge data. Fetal death risks were determined on a "per-pair-at-risk" basis., Results: While the proportion of twins born at 24-31 weeks remained stable at 8%, the proportion born at 32-36 weeks increased from 28% to 48%, and the proportion at 37-42 weeks declined from 64% to 44% (P<0.0001). Controlling individually for a variety of factors, such as maternal age, race, parity, and mode of delivery did not diminish the highly significant trend of increasing preterm births (P<0.0001 for each). Twin pair neonatal mortality decreased significantly through time (P<0.0001); however, the rate of pairs with one or both infants requiring oxygen or ventilation increased significantly through time (P<0.0001). Fetal death risks declined for term twins., Conclusions: The proportion of twins born at 32-36 weeks' gestation has increased over time, along with requirement for respiratory support. Twin pair mortality decreased from 1980 to 2005.

The earliest stages of animal development occur without the benefit of zygotic transcription. The absence of transcription necessitates that all changes in the levels of specific proteins must be controlled by post-transcriptional mechanisms, such as the regulated translation of stored maternal mRNAs. One of the major challenges to investigating translational mechanisms is the availability of reliable methods for assaying the translational state of specific mRNAs. The most definitive assay of an mRNA's translational state is polyribosome association; mRNAs actively translated are engaged with polyribosomes while mRNAs translationally repressed are not. While linear gradient centrifugation is commonly used to purify polyribosomes from a wide variety of cell types in different organisms, the isolation of polyribosomes from Xenopus oocytes, eggs and embryos presents some unique challenges. Here we detail the methodology for the isolation and analysis of polyribosomes from Xenopus oocytes, eggs and embryos using step gradient centrifugation. We present detailed protocols, describe the critical controls and provide several examples to guide the interpretation of experimental results regarding the translational state of specific mRNAs.

The cell cycle regulator cyclin E1 is aberrantly expressed in a variety of human cancers. In breast cancer, elevated cyclin E1 correlates with poor outcome, as do high cytoplasmic levels of the stress-induced RNA-binding protein human antigen R (HuR). We showed previously that increased cytoplasmic HuR elevates cyclin E1 in MCF-7 breast cancer cells by stabilizing its mRNA. We show here that cold-inducible RNA-binding protein (CIRP) co-regulates cyclin E1 with HuR in breast cancer cells. CIRP had been shown to interact with HuR in Xenopus laevis oocytes and to be decreased in endometrial cancer. To investigate if human CIRP and HuR co-regulate cyclin E1, HuR and CIRP levels were altered in MCF-7 cells and effects on cyclin E1 assessed. Altering HuR expression resulted in a reciprocal change in CIRP expression, while altering CIRP expression resulted in corresponding changes in HuR and cyclin E1 expression. CIRP and HuR co-precipitated in the presence of RNA and CIRP enhanced HuR binding to the cyclin E1 mRNA and increased cyclin E1 mRNA stability. CIRP co-localized with HuR predominantly in the nucleus, but also in discrete cytoplasmic foci identified as stress granules (SGs). CIRP overexpression increased the number of HuR-containing SGs, while its knockdown decreased them. Our results suggest that CIRP positively regulates HuR, ultimately resulting in increased protein synthesis of at least one of its targets.

Background: The neural crest is a unique population of cells that arise in the vertebrate ectoderm at the neural plate border after which they migrate extensively throughout the embryo, giving rise to a wide range of derivatives. A number of proteins involved in neural crest development have dynamic expression patterns, and it is becoming clear that ubiquitin-mediated protein degradation is partly responsible for this., Results: Here we demonstrate a novel role for the F-box protein Cdc4/Fbxw7 in neural crest development. Two isoforms of Xenopus laevis Cdc4 were identified, and designated xCdc4alpha and xCdc4beta. These are highly conserved with vertebrate Cdc4 orthologs, and the Xenopus proteins are functionally equivalent in terms of their ability to degrade Cyclin E, an established vertebrate Cdc4 target. Blocking xCdc4 function specifically inhibited neural crest development at an early stage, prior to expression of c-Myc, Snail2 and Snail., Conclusions: We demonstrate that Cdc4, an ubiquitin E3 ligase subunit previously identified as targeting primarily cell cycle regulators for proteolysis, has additional roles in control of formation of the neural crest. Hence, we identify Cdc4 as a protein with separable but complementary functions in control of cell proliferation and differentiation.

We show that microRNA-427 (miR-427) mediates the rapid deadenylation of maternal mRNAs after the midblastula transition (MBT) of Xenopus laevis embryogenesis. By MBT, the stage when the embryonic cell cycle is remodeled and zygotic transcription of mRNAs is initiated, each embryo has accumulated approximately 10(9) molecules of miR-427 processed from multimeric pri-miR-427 transcripts synthesized after fertilization. We demonstrate that the maternal mRNAs for cyclins A1 and B2 each contain a single miR-427 target sequence, spanning less than 30 nucleotides, that is both necessary and sufficient for deadenylation, and that inactivation of miR-427 leads to stabilization of the mRNAs. Although this deadenylation normally takes place after MBT, exogenous miRNAs produced prematurely in vivo can promote deadenylation prior to MBT, indicating that turnover of the maternal mRNAs is limited by the amount of accumulated miR-427. Injected transcripts comprised solely of the cyclin mRNA 3' untranslated regions or bearing a 5' ApppG cap undergo deadenylation, showing that translation of the targeted RNA is not required. miR-427 is not unique in promoting deadenylation, as an unrelated miRNA, let-7, can substitute for miR-427 if the reporter RNA contains an appropriate let-7 target site. We propose that miR-427, like the orthologous miR-430 of zebrafish, functions to down-regulate expression of maternal mRNAs early in development.

MicroRNAs (miRNAs) are a class of naturally occurring, small, non-coding RNAs that control gene expression during development,normal cell function and disease. Although there is emerging evidence that some miRNAs can function as oncogenes or tumor suppressors, there is limited understanding of the role of miRNAs in cancer. In this study, we observed that the expression of miR-125a was inversely correlated with HuR expression in several different breast carcinoma cell lines. HuR is a stress-induced RNA binding protein whose expression is elevated or localization perturbed in several different cancers. Increased cytoplasmic localization of HuR is a prognostic marker in breast cancer. Real time PCR and gene reporter assays indicated that HuR was translationally repressed by miR-125a. Re-establishing miR-125a expression in breast cancer cells decreased HuR protein level and inhibited cell growth. Using MCF-7 breast cancer cells, we further clarified that miR-125a inhibited cell growth via a dramatic suppression of cell proliferation and promotion of apoptosis.In addition, cell migration was also inhibited by miR-125a overexpression. Importantly, the repression of cell proliferation and migration engendered by miR-125a was partly rescued by HuR re-expression. Our results suggest that miR-125a may function as a tumor suppressor for breast cancer, with HuR as a direct and functional target.

In Xenopus embryos, maternal cyclins drive the first 12 cell divisions after which several cyclins are terminally degraded, including cyclin B2. Cyclin B2 disappearance is due to transcription-mediated mRNA deadenylation at the midblastula transition, when transcription initiates and the cell cycle lengthens. To further define the mechanism, we characterized proteins capable of binding cyclin B2 3'UTR. We show that ElrA and AUF1 compete for binding to regions containing cytoplasmic polyadenylation elements (CPEs), with AUF1 binding increasing at the midblastula transition. Deletion of both CPEs abrogates polyadenylation but has no effect on deadenylation or binding of ElrA or AUF1. Overexpression of ElrA or AUF1 does not alter cyclin B2 mRNA stability. These results show that ElrA and AUF1 bind to cyclin B2 mRNA independent of CPEs and function by binding other elements.

The early cell divisions of Xenopus laevis and other metazoan embryos occur in the presence of constitutively high levels of the cell cycle regulator cyclin E1. Upon completion of the 12th cell division, a time at which many maternal proteins are downregulated by deadenylation and destabilization of their encoding mRNAs, maternal cyclin E1 protein is downregulated while its mRNA is polyadenylated and stable. We report here that stable polyadenylation of cyclin E1 mRNA requires three cis-acting elements in the 3' untranslated region; the nuclear polyadenylation sequence, a contiguous cytoplasmic polyadenylation element and an upstream AU-rich element. ElrA, the Xenopus homolog of HuR and a member of the ELAV gene family binds the cyclin E1 3'UTR with high affinity. Deletion of these elements dramatically reduces the affinity of ElrA for the cyclin E1 3'UTR, abolishes polyadenylation and destabilizes the mRNA. Together, these findings provide compelling evidence that ElrA functions in polyadenylation and stabilization of cyclin E1 mRNA via binding these elements.

Many cancers overexpress cyclin E1 and its tumor-specific low molecular weight (LMW) isoforms. However, the mechanism of cyclin E1 deregulation in cancers is still not well understood. We show here that the mRNA-binding protein HuR increases cyclin E1 mRNA stability in MCF-7 breast carcinoma cells. Thus, mRNA stabilization may be a key event in the deregulation of cyclin E1 in MCF-7 cells. Compared with MCF10A immortalized breast epithelial cells, MCF-7 cells overexpress full-length cyclin E1 and its LMW isoforms and exhibit increased cyclin E1 mRNA stability. Increased mRNA stability is associated with a stable adenylation state and an increased ratio of cytoplasmic versus nuclear HuR. UV cross-link competition and UV cross-link immunoprecipitation assays verified that HuR specifically bound to the cyclin E1 3'-untranslated region. Knockdown of HuR with small interfering RNA (siRNA) in MCF-7 cells decreased cyclin E1 mRNA half-life (t(1/2)) and its protein level: a 22% decrease for the full-length isoforms and 80% decrease for the LMW isoforms. HuR siRNA also delayed G(1)-S phase transition and inhibited MCF-7 cell proliferation, which was partially recovered by overexpression of a LMW isoform of cyclin E1. Overexpression of HuR in MCF10A cells increased cyclin E1 mRNA t(1/2) and its protein level. Taken together, our data show that HuR critically contributes to cyclin E1 overexpression and its growth-promoting function, at least in part by increasing cyclin E1 mRNA stability, which provides a new mechanism of cyclin E1 deregulation in breast cancer.

In Xenopus laevis embryos, cyclin E protein remains constitutively high throughout the first 12 cell cycles following fertilization until the onset of the midblastula transition (MBT) (after the 12(th) cell cycle) when it undergoes a dramatic reduction. The disappearance of cyclin E at the MBT occurs independently of active cell cycle progression, zygotic transcription, protein synthesis and the nuclear to cytoplasmic ratio. This has suggested that cyclin E is part of an autonomous maternal timer that regulates the onset of the MBT. To determine how constitutively high levels of cyclin E are maintained prior to the MBT and to investigate if the reduction in cyclin E protein affects the timing of the MBT, we have knocked down endogenous cyclin E mRNA using an N,N-diethyl-ethylene-diamine modified antisense oligonucleotide targeted to its open reading frame. We report that maintenance of high levels of cyclin E protein before the MBT is due to a balance between ongoing translation and proteolytic degradation. In support of our antisense experiments, polysome analysis demonstrates that cyclin E mRNA is associated with the translated fraction prior to the MBT. Furthermore, knockdown of cyclin E was not associated with defects in the timing of developmental events. Our data suggests that cyclin E is not required for the later cell cycles of embryonic development and that the pathway effecting downregulation of cyclin E rather then cyclin E degradation itself may be part of a maternal timer that affects the onset of the MBT.

Objective: To determine whether second-born twins (B) have higher morbidity and mortality than first-born twins (A), using a paired analysis., Study Design: We conducted a retrospective analysis of birth certificates and fetal and infant death certificates for 5138 twin pairs selected from those born in Washington State from 1989 to 2001. Twin A was vertex and delivered vaginally. Pairs were not size-discordant (< 20%) and had no malformations. Matched-pair odds ratios were calculated., Results: Twin B had more fetal distress (OR=6.0) and more low 5-min Apgar scores (OR=2.1) than Twin A, except at short delivery intervals. Pairs had relatively high rates of combined vaginal plus cesarean deliveries at delivery intervals 15 min., Conclusion: If prompt vaginal delivery of Twin B does not occur, the benefits of vaginal delivery for Twin A might not outweigh the risks of distress and low Apgar scores in Twin B and vaginal plus cesarean delivery for the mother.

Deregulation of gene expression is a hallmark of the cancer cell. Acquiring a new profile of expressed proteins may enable the cell to re-enter the cell cycle, or give them a growth or motility advantage over "normal cells". An efficient and rapid way to alter gene expression is via regulation of mRNAs already transcribed. Modifications of mRNA stability and/or translational efficiency are increasingly reported in cancer. mRNA stability and translation are controlled through a complex network of RNA/protein interactions involving recognition of specific target mRNAs by RNA-BPs. We review how alterations in regulatory sequences, RNA-BPs, or in upstream signalling pathways affect the stability and/or translational efficiency of mRNAs encoding proto-oncogenes, cytokines, cell cycle regulators and other regulatory proteins to promote tumorigenesis and cancer progression. A more thorough understanding of post-transcriptional mechanisms such as these will enable the design and development of specific therapies based on modulating the translation or stability of specific mRNAs., (Copyright 2004 Elsevier SAS)

Cyclin mRNAs are unstable in the adult cell cycle yet are stable during the first 12 cell divisions in Xenopus laevis. We recently reported that cyclin A1 and B2 maternal mRNAs are deadenylated upon completion of the 12th division (Audic et al. [2001] Mol. Cell Biol. 21:1662-1671). Deadenylation is mediated by the 3' untranslated region (UTR) of the mRNA and precedes the terminal disappearance of the cyclin proteins, with both processes requiring zygotic transcription. The purpose of the current study was (1) to ask whether deadenylation leads to translational repression and/or destabilization of endogenous cyclin A1 and B2 mRNAs, and (2) to further characterize the regulatory sequences required. We show that zygote-driven deadenylation leads to translational repression and mRNA destabilization. A 99-nucleotide region of the 3'UTR of the cyclin A1 mRNA mediates both deadenylation and destabilization. Surprisingly, two AU-rich consensus elements within this region are dispensable for this activity. These results suggest that zygote-dependent deadenylation, translational repression, and mRNA destabilization by means of novel 3'UTR elements contribute to the disappearance of maternal cyclins. They also suggest that translational control of cyclins may play a role in the transition to the adult cell cycle. These data concur with previous studies in Drosophila showing that zygote-mediated degradation of maternal cdc25 mRNA may be a general mechanism whereby transition to the adult cell cycle proceeds., (Copyright 2002 Wiley-Liss, Inc.)

Objective: The purpose of this study was to determine whether size-discordant twin pairs have worse perinatal mortality and neonatal morbidity rates than nondiscordant pairs and whether the smaller twins of discordant pairs have worse perinatal outcomes than the larger twins., Study Design: We conducted a population-based, retrospective analysis of linked birth certificates and fetal and infant death certificates for 9590 twin pairs who were born in the state of Washington from 1987 through 1999. The Cochran-Mantel-Haenszel test, Student t test, and McNemar test were among the tests used to assess statistical significance., Results: Discordant twin pairs had higher rates of perinatal mortality, neonatal mortality, and 5-minute Apgar scores of <7, even after stratification by gestational age. Discordant pairs had lower pair weights at each gestational age and were more likely to include small-for-gestational-age infants. Compared with the larger twins, the smaller twins of discordant pairs had higher rates of perinatal mortality., Conclusion: Discordant pairs had worse perinatal outcomes within each gestational age category.

At the midblastula transition, the Xenopus laevis embryonic cell cycle is remodeled from rapid alternations between S and M phases to become the complex adult cell cycle. Cell cycle remodeling occurs after zygotic transcription initiates and is accompanied by terminal downregulation of maternal cyclins A1 and B2. We report here that the disappearance of both cyclin A1 and B2 proteins is preceded by the rapid deadenylation of their mRNAs. A specific mechanism triggers this deadenylation. This mechanism depends upon discrete regions of the 3' untranslated regions and requires zygotic transcription. Together, these results strongly suggest that zygote-dependent deadenylation of cyclin A1 and cyclin B2 mRNAs is responsible for the downregulation of these proteins. These studies also raise the possibility that zygotic control of maternal cyclins plays a role in establishing the adult cell cycle.

Objective: The aim of this study was to determine the gestational age at delivery associated with the lowest rates of perinatal mortality, respiratory distress syndrome, and long hospital stays among twins, with pair rates used to account for both infants in each twin pregnancy., Study Design: We conducted a population-based retrospective study that analyzed linked birth certificates, fetal and infant death certificates, and hospital discharge data for 8150 twin pairs born in Washington State during 1987 through 1997. The chi2 or Fisher exact test was used to assess the statistical significance., Results: The nadirs of perinatal mortality rate, respiratory distress syndrome incidence, and long hospital stay rate were seen at delivery dates of 39, 40, and 38 weeks' gestation, respectively. Restriction to pairs delivered vaginally without the induction of labor revealed that the perinatal mortality rate was lowest for delivery at 37 weeks' gestation, the gestational age at which the highest numbers of such spontaneously timed pairs were born., Conclusion: Induction of labor should be routinely considered for twins at 37 to 38 weeks' gestation.

A subset of adult peripheral blood leukocytes functions as endothelial cell progenitors called angioblasts. They can incorporate into the vasculature in animal models of neovascularization and accelerate the restoration of blood flow to mouse ischemic limbs. Earlier reports suggested that CD34-expressing (CD34+) but not CD34+ cell-depleted (CD34-) leukocytes can differentiate into endothelial cells (EC) in vitro and in vivo. Recent findings suggest that CD14+ cells, which are typically CD34-, also have angioblast-like properties in vitro. To determine the identity of angioblasts, the potential of CD34+, CD34-, CD34- CD14+, and CD34- CD14- cells to produce EC was compared. We show that a subset of monocyte (CD34- CD14+)-enriched cells can take on an EC-like phenotype in culture, but that the EC-like cells also express dendritic cell antigens. These findings suggest that monocytes differentiate into macrophages, dendritic cells, or EC depending on environmental cues. The data also demonstrate that angioblasts are more abundant in the blood than previously thought. Finally, we demonstrate that CD34- and CD34- CD14+ cells incorporate into the endothelium of blood vessels in mouse ischemic limbs. However, incorporation of these cells requires co-injection with CD34+ cells, indicating that leukocyte-leukocyte interactions may play a critical role in governing angioblast behavior in vivo.

Emerging data suggest that current strategies for the treatment of spinal cord injury might be improved or augmented by spinal cord grafts of neural cells, and it is possible that grafted neurons might have therapeutic potential. Thus, here we have summarized recent studies of the neurobiology of clonal human (NT2N) neurons grafted into spinal cord of immunodeficient athymic nude mice. Postmitotic human NT2N neurons derived in vitro from an embryonal carcinoma cell line (NT2) were transplanted into spinal cord of neonatal, adolescent and adult nude mice where they became integrated into the host gray and white matter, did not migrate from the graft site, and survived for > 15 months after implantation. The neuronal phenotype of the grafted NT2N cells was similar in gray and white matter regardless of host age at implantation, and some of the processes extended by the transplanted NT2N neurons became ensheathed by oligodendrocytes. However, there were consistent differences between NT2N processes traversing white versus gray matter. Most notably, NT2N processes with a trajectory in white matter extended over much longer distances (some for > 2 cm) than those confined to gray matter. Thus, NT2N neurons grafted into spinal cord of nude mice integrated into gray as well as white matter, where they exhibited and maintained the morphological and molecular phenotype of mature neurons for > 15 months after implantation. Also, the processes extended by grafted NT2N neurons differentially responded to cues restricted to gray versus white matter. Further insight into the neurobiology of grafted human NT2N neurons in the normal and injured spinal cord of experimental animals may lead to novel and more effective strategies for the treatment of spinal cord injury.

To investigate host effects on grafts of pure, postmitotic, human neurons, we assessed the morphologic and molecular phenotype of purified NTera2N (NT2N, hNT) neurons implanted into the spinal cord of athymic nude mice. NT2N neurons were implanted into both spinal cord gray matter and white matter of neonatal, adolescent, and adult mice and were evaluated at postimplantation times up to 15 months. NT2N neurons remained at the implantation site and showed process integration into all host areas, and each graft exhibited similar phenotypic features regardless of location or host age at implantation. Evidence of host oligodendrocyte ensheathment of NT2N neuronal processes was seen, and grafted NT2N neurons acquired and maintained the morphologic and molecular phenotype of mature neurons. The microenvironments of host gray matter and white matter appear to exert differential effects on implanted neuronal processes, because consistent differences were noted in the morphologies of graft processes extending into white matter versus gray matter. NT2N processes extended for long distances (>2 cm) within white matter, whereas NT2N processes located within gray matter had shorter trajectories. This suggests that NT2N neurons integrate similarly into spinal cord gray matter and white matter, but they extend processes that respond differentially to gray matter and white matter cues. Further studies of the model system described here may identify the host molecular signals that support and direct integration of grafted human neurons as well as the outgrowth of their processes in the nervous system., (Copyright 1999 Wiley-Liss, Inc.)

The formation of functional synapses is a late milestone of neuronal differentiation. The establishment of functional synapses can be used to assess neuronal characteristics of different cell lines. In the present study, we examined the in vitro conditions that influence the ability of human neurons derived from the NT2 cell line (NT2N neurons) to establish synapses. The morphologic, immunologic, and electrophysiologic characteristics of these synapses was examined. In the absence of astrocytes, NT2N neurons rarely formed synapses and their action potentials were weak and uncommon. In contrast, when plated on primary astrocytes, NT2N neurons were able to form both glutamatergic excitatory (71%) and GABAergic inhibitory (29%) functional synapses whose properties (kinetics, ion selectivity, pharmacology, and ultrastructure) were similar to those of synapses of neurons in primary cultures. In addition, coculture of NT2N neurons with astrocytes modified the morphology of the neurons and extended their in vitro viability to more than 1 year. Because astrocyte-conditioned medium did not produce these effects, we infer that direct contact between NT2N neurons and astrocytes is required. These results suggest that NT2N neurons are similar to primary neurons in their synaptogenesis and their requirement for glial support for optimal survival and maturation. This system provides a model for further investigations into the neurobiology of synapses formed by human neurons.

During Xenopus development, the early cell cycles consist of rapid oscillations between DNA synthesis and mitosis until completion of the 12th mitotic division. Then the cycle lengthens and becomes asynchronous, zygotic transcription begins, and G phases are established, a period known as the midblastula transition (MBT). Some aspects of the MBT, such as zygotic transcription, depend on acquisition of a threshold nuclear to cytoplasmic (N/C) ratio, whereas others, such as maternal cyclin E degradation, are independent of nuclear events and appear to be controlled by an autonomous maternal timer. To investigate the function of cyclin E during the early cycles, cyclin E/Cdk2 kinase activity was specifically inhibited in fertilized eggs by a truncated form of the Xenopus Cdk inhibitor, Xic1 (Delta34Xic1). Delta34Xic1 caused lengthening of the embryonic cell cycles that correlated with increased levels of mitotic cyclins. However, DNA synthesis was not inhibited. Several hallmarks of the MBT were delayed for several hours in Delta34Xic1-injected embryos, including the disappearance of cyclins E and A, the initiation of zygotic transcription, and the reappearance of phosphotyrosine on Cdc2. In both control and Delta34Xic1-injected embryos, cyclin E was degraded after the 12th mitotic division as zygotic transcription began, but experiments with alpha-amanitin show that cyclin E degradation is not dependent on zygotic transcription. Thus, the length of the early cycles and the timing of maternal cyclin degradation depend upon cyclin E/Cdk2 activity. Neither oscillations in cyclin E/Cdk2 activity during the early cycles nor the disappearance of cyclin E at the MBT were dependent on protein synthesis. These data suggest that cyclin E/Cdk2 is directly linked to an autonomous maternal timer that drives the early embryonic cell cycles until the MBT.

Male canaries (Serinus canaria) produce most of their song syllable types on the left side of the syrinx, the two-sided vocal organ. Female canaries treated with testosterone propionate begin to sing in a male fashion, a behavior seldom seen in untreated females. To learn whether syringeal dominance occurs in testosterone-treated female canaries, we deactivated either the right or the left side of the syrinx by severing the tracheosyringeal nerve on that side. Recordings of the birds' songs were made before and after the nerve cut, and song analysis was based upon visual comparisons of sound spectrograms. Pre- and post-operative syllable types were identified by their frequency structures and repetition rates. Six of eight birds displayed clear left dominance as assessed by nerve cuts; therefore, we concluded that most, but not all, female canaries generate the majority of their syllable types with the left syrinx.

We have characterized a human embryonal carcinoma cell line (NTera-2 or NT2 cells) that is transfectable and capable of differentiating into postmitotic neuron-like cells (NT2N cells) following treatment with retinoic acid in order to identify a human neuronal cell line that might serve as a "platform" for gene therapy of human neurological diseases. Studies of NT2N cells transplanted into the brain or spinal cord of immunecompetent and immunodeficient rodents show that NT2N cells integrate into the host central nervous system (CNS) and establish the molecular and structural polarity of authentic neurons in vivo. Further, grafted NT2N cells acquire the molecular phenotype of fully mature neurons within 6 months postimplantation and the grafts survive > 1 year in immunodeficient mice without reverting to a neoplastic state. Although grafts of the retinoic acid-naive NT2 cells can form lethal tumors in the CNS, these cells differentiate into postmitotic neuron-like cells and do not form tumors when the grafts are confined to the caudoputamen. Based on the studies reviewed here, we conclude that grafted NT2N cells could serve as a suitable platform for the delivery of exogenous proteins into the CNS for gene therapy of human nervous system diseases.

The relationship between the motor and acoustic similarity of song was examined in brown thrashers (Toxostoma rufum) and grey catbirds (Dumetella carolinensis) (family Mimidae), which have very large song repertoires and sometimes mimic other species. Motor similarity was assessed by cross correlation of syringeal airflows and air sac pressures that accompany sound production. Although most syllables were sung only once in the song analyzed, some were repeated, either immediately forming a couplet, or after a period of intervening song, as a distant repetition. Both couplets and distant repetitions are produced by distinctive, stereotyped motor patterns. Their motor similarity does not decrease as the time interval between repetitions increases, suggesting that repeated syllables are stored in memory as fixed motor programs. The acoustic similarity between nonrepeated syllables, as indicated by correlation of their spectrograms, has a significant positive correlation with their motor similarity. This correlation is weak, however, suggesting that there is no simple linear relationship between motor action and acoustic output and that similar sounds may sometimes be produced by different motor mechanisms. When compared without regard to the sequence in which they are sung, syllables paired for maximum spectral similarity form a continuum with repeated syllables in terms of their acoustic and motor similarity. The prominence of couplets in the "syntax" of normal song is enhanced by the dissimilarity of successive nonrepeated syllables that make up the remainder of the song.

We report here the first extensive in vivo study of cell cycle regulation in the Xenopus embryo. Cyclin A1, B1, B2, and E1 levels, Cdc2 and Cdk2 kinase activity, and Cdc25C phosphorylation states were monitored during early Xenopus embryonic cell cycles. Cyclin B1 and B2 protein levels were high in the unfertilized egg, declined upon fertilization, and reaccumulated to the same level during the first cell cycle, a pattern repeated during each of the following 11 divisions. Cyclin A1 showed a similar pattern, except that its level was lower in the egg than in the cell cycles after fertilization. Cyclin B1/Cdc2 kinase activity oscillated, peaking before each cleavage, and Cdc25C alternated between a highly phosphorylated and a less phosphorylated form that correlated with high and low cyclin B1/Cdc2 kinase activity, respectively. Unlike the mitotic cyclins, the level of cyclin E1 did not oscillate during embryogenesis, although its associated Cdk2 kinase activity cycled twice for each oscillation of cyclin B1/Cdc2 activity, consistent with a role for cyclin E1 in both S-phase and mitosis. Although the length of the first embryonic cycle is regulated by both the level of cyclin B and the phosphorylation state of Cdc2, cyclin accumulation alone was rate-limiting for later cycles, since overexpression of a mitotic cyclin after the first cycle caused cell cycle acceleration. The activity of Cdc2 closely paralleled the accumulation of cyclin B2, but cell cycle acceleration caused by cyclin B overexpression was not associated with elevation of Cdc2 activity to higher than metaphase levels. Tyrosine phosphorylation of Cdc2, absent during cycles 2-12, reappeared at the midblastula transition coincident with the disappearance of cyclin E1. Cyclin A1 disappeared later, at the beginning of gastrulation. Our results suggest that the timing of the cell cycle in the Xenopus embryo evolves from regulation by accumulation of mitotic cyclins to mechanisms involving periodic G1 cyclin expression and inhibitory tyrosine phosphorylation of Cdc2.

Mitogen-activated protein kinase (MAPK) is one of the protein kinases activated during meiotic maturation of Xenopus laevis oocytes. The c-Mosxe protein kinase, which has been shown to be sufficient to promote germinal vesicle breakdown (GVBD) in meiosis I, can directly activate MAP kinase kinase in vitro and leads to the activation of MAPK in vivo. Recently we have shown that constitutively activated MAPK induces metaphase arrest when injected into one blastomere of a two-cell embryo. This arrest mimics the natural arrest of vertebrate unfertilized eggs in second meiotic metaphase due to cytostatic factor and c-Mosxe activity. We show here that microinjection of constitutively activated thiophosphorylated MAPK into resting oocytes is able to activate maturation-promoting factor (MPF) and promote GVBD. These results strongly support the hypothesis that MAPK plays an important role in the pathway that links c-Mosxe to the activation of MPF.

In brown thrashers (Toxostoma rufum) and grey catbirds (Dumetella carolinensis) neither side of the syrinx has a consistently dominant role in song production. During song, the two sides operate independently, but in close cooperation with each other and with the respiratory muscles which are capable of adjusting expiratory effort to maintain a constant rate of syringeal airflow despite sudden changes in syringeal resistance. Phonation is frequently switched from one side of the syrinx to the other, both between syllables and within a syllable. When both sides of the syrinx produce sound simultaneously, their respective contributions are seldom harmonically related. The resulting "two-voice" syllables sometimes contain difference tones with prominent sinusoidal amplitude modulation (AM). Rarely, both sides simultaneously produce the same sound. In general, however, the frequency range of sound contributed by the right syrinx is higher than that of the left syrinx. The right syrinx is also primarily responsible for producing a rapid cyclical amplitude modulation which is a characteristic feature of some syllables. This kind of AM is generated by either repetitive brief bursts of sound from the right side that modulate the amplitude of a continuous sound arising on the left side or cyclically opening the right syrinx, allowing unmodulated expiratory air to bypass the phonating left side.

MAP kinase (MAPK) is activated in animal cap explants from Xenopus embryos in response to mesoderm induction by FGF. This activation is rapid, appearing within 1 min of treatment with FGF, and prolonged, lasting for at least 2 hr. By immunoblot analysis, this activation of MAPK is coupled with an electrophoretic shift to the slowly migrating, phosphorylated form of MAPK. Activin-stimulated mesoderm induction also results in the activation of MAPK, but only upon prolonged exposure. However, activin can potentiate the activation of MAPK by FGF as early as 1 min after administration. These findings suggest that MAPK is involved in the early signaling events of FGF-mediated mesoderm induction, and this involvement is modulated by other mesoderm inducers such as activin.

The emergence of avian satellite cells during development has been studied using markers that distinguish adult from fetal cells. Previous studies by us have shown that myogenic cultures from fetal (Embryonic Day 10) and adult 12-16 weeks) chicken pectoralis muscle (PM) each regulate expression of the embryonic isoform of fast myosin heavy chain (MHC) differently. In fetal cultures, embryonic MHC is coexpressed with a ventricular MHC in both myocytes (differentiated myoblasts) and myotubes. In contrast, myocytes and newly formed myotubes in adult cultures express ventricular but not embryonic MHC. In the current study, the appearance of myocytes and myotubes which express ventricular but not embryonic MHC was used to determine when adult myoblasts first emerge during avian development. By examining patterns of MHC expression in mass and clonal cultures prepared from embryonic and posthatch chicken skeletal muscle using double-label immunofluorescence with isoform-specific monoclonal antibodies, we show that a significant number of myocytes and myotubes which stain for ventricular but not embryonic MHC are first seen in cultures derived from PM during fetal development (Embryonic Day 18) and comprise the majority, if not all, of the myoblasts present at hatching and beyond. These results suggest that adult type myoblasts become dominant in late embryogenesis. We also show that satellite cell cultures derived from adult slow muscle give results similar to those of cultures derived from adult fast muscle. Cultures derived from Embryonic Day 10 hindlimb form myocytes and myotubes that coexpress ventricular and embryonic MHCs in a manner similar to cells of the Embryonic Day 10 PM. Thus, adult and fetal expression patterns of ventricular and embryonic MHCs are correlated with developmental age but not muscle fiber type.

We compared the expression of myosin heavy chains in myogenic cultures prepared from fetal (embryonic Day 10) and adult (12-16 weeks) chicken pectoralis muscle using immunofluorescence with isoform-specific monoclonal antibodies. We found that the majority of fetal myocytes (differentiated myoblasts) and myotubes coexpressed ventricular and embryonic myosin heavy chains in culture. Also, when fetal cells were plated at a clonal density most clones coexpressed both ventricular and embryonic isoforms. In contrast, all adult myocytes and newly formed adult myotubes expressed just ventricular myosin, whether plated at mass or clonal densities. Within 12-24 hr of the onset of fusion, adult myotubes began to express embryonic myosin as well. Eventually, the majority of adult myotubes coexpressed both ventricular and embryonic myosin. The delay of embryonic myosin expression until after fusion was also seen in passaged adult myoblasts and in myoblasts isolated from regenerating adult muscle. The expression of embryonic myosin can be abolished by inhibiting fusion with EGTA in adult but not in fetal cultures. We conclude that both fetal and adult myotubes express ventricular and embryonic myosins but only fetal myocytes express the embryonic isoform prior to fusion. This difference in the regulation of embryonic myosin expression between fetal and adult myoblasts supports the hypothesis that these cells may represent two distinct populations of myogenic precursors.

The canary (Serinus canaria) vocal organ, the syrinx, has two separate sound sources, one in the cranial end of each bronchus. Previous investigations of whether song syllables are produced unilaterally or bilaterally have provided two contradictory results, as one researcher suggested that almost all syllables are produced by the left side of the syrinx alone, whereas another researcher suggested that both sides contribute similarly to all syllables. Our experiments, which involved unilateral bronchus plugging followed later by denervation of the ipsilateral syringeal muscles, attempted to resolve this disagreement. The males with right bronchus plugs, singing on the left side of the syrinx alone, produced nearly normal songs, whereas the birds with left bronchus plugs, singing on the right side, sang quite poorly. Interpretation of these data is difficult because it is not clear how syringeal function would be affected if the airflow rate through the intact side is increased above normal, nor is it known if the bird can compensate for bronchus occlusion. Nonetheless, we suggest that in male canaries most syllables are normally sung by the left side alone, with some syllables being produced by the right side alone and some being sung by both sides together. Right nerve section had little effect on the right-bronchus-plugged males' ability to sing, but the repertoires of the left-plugged males were altered after left nerve section, indicating the possibility that signals carried by the left nerve exert an influence on the contralateral side.

We describe a simple technique for maintaining highly contractile long-term chicken myogenic cultures on Matrigel, a gel composed of basement membrane components extracted from the Engelbreth-Holm-Swarm mouse tumor. Cultures grown on Matrigel consist of three-dimensional multilayers of cylindrical, contracting myotubes which endure for at least 60 d without myotube detachment. A Matrigel substrate increases the initial plating efficiency but does not effect cell proliferation. Large-scale differentiation in cultures maintained on Matrigel is delayed by 1 to 2 d, compared to cultures grown on gelatin-coated dishes. Long-term maintenance on Matrigel also results in increased expression of the neonatal and adult fast myosin heavy chain isoforms. Culturing of cells on a Matrigel substrate could thus facilitate the study of later events of in vitro myogenesis.

Elaborate respiratory patterns accompany song production in male canaries (Serinus canaria). To learn how such patterns arise, electromyographic activity was measured in the expiratory muscles in the abdomen. Most song phrases are accompanied either by mini-breaths (when syllable repetition rates are 2 to 27/sec) or by pulsatile expiration (when syllable repetition rates are 30 to 38/sec). In both cases there is a one-to-one correspondence between bursts of expiratory muscle electrical activity and song syllables. Phrases with syllable repetition rates of 62-70/sec, which are rare, are accompanied by expiratory airflow that may be either pulsatile or continuous. The expiratory muscles are active throughout such phrases, suggesting that the muscles of the vocal organ, the syrinx, are responsible for producing separate notes. Thus, at rates up to 38/sec, the abdominal muscles of canaries contract briefly for the production of each song syllable.