Zinc regulates reactive oxygen species generation in platelets.

Vascular complications resulting from atherosclerosis development are a major cause of death. Reactive oxygen species (ROS) are produced by platelets during activation, and have been demonstrated to positively regulate platelet activatory responses. Zn2+ is also an important hemostatic cofactor in platelets, acting both as a platelet agonist and second messenger. Whilst the effect of Zn2+-dependent signaling mechanisms on ROS production in nucleated cells has been demonstrated, comparable roles in platelets have yet to be investigated. In this study we investigated the relationship between fluctuations in cytosolic Zn2 [Zn2+]i and platelet ROS production. Agonist-evoked ROS production, GSH levels and GPx activity are abrogated in platelets treated with the Zn2+-chelator, TPEN. Conversely, increasing platelet [Zn2+]i using Zn2+ ionophores potentiated ROS generation and decreased GSH levels and GPx activity. Zn2+-dependent ROS production was sensitive to pretreatment with DPI or mitoTEMPO, NADPH oxidase and mitochondria inhibitors respectively. Increasing [Zn2+]i resulted in increases of Erk1/2 and JNK phosphorylation. Our data are consistent with a functional association between [Zn2+]i and ROS production in platelets that could influence thrombus formation in a clinical context.

Post-combustion carbon dioxide capture cost reduction to 2030 and beyond.

Post-combustion CO2 capture (PCC) can be achieved using a variety of technologies. Importantly it is applicable to a wide range of processes and may also be retrofitted in certain cases. This paper covers the use of PCC for low carbon power generation from new natural gas combined cycle (NGCC) plants that are expected to be built in the UK in the 2020s and 2030s and that will run into the 2050s. Costs appear potentially comparable with other low carbon and controllable generation sources such as nuclear or renewables plus storage, especially with the lower gas prices that can be expected in a carbon-constrained world. Non-fuel cost reduction is still, however, desirable and, since CO2 capture is a new application, significant potential is likely to exist. For the NGCC+PCC examples shown in this paper, moving from 'first of a kind' (FOAK) to 'nth of a kind' (NOAK) gives significant improvements through both reduced financing costs and capital cost reductions. To achieve this the main emphasis needs to be on 'commercial readiness', rather than on system-level 'technical readiness', and on improvements through innovation activities, supported by underpinning research, that develop novel sub-processes; this will also maintain NOAK status for cost-effective financing. Feasible reductions in the energy penalty for PCC capture have much less impact, reflecting the inherently high levels of efficiency for modern NGCC+PCC plants.

Recognizing that it is part and parcel of what they do: teaching palliative care to medical students in the UK.

In their first year of work, newly qualified doctors will care for patients who have palliative care needs or who are dying, and they will need the skills to do this throughout their medical career. The General Medical Council in the United Kingdom has given clear recommendations that all medical students should receive core teaching on relieving pain and distress together with caring for the terminally ill. However, medical schools provide variable amounts of this teaching; some are able to deliver comprehensive programmes whilst others deliver very little. This paper presents the results of a mixed methods study which explored the structure and content of palliative care teaching in different UK medical schools, and revealed what coordinators are trying to achieve with this teaching. Nationally, coordinators are aiming to help medical students overcome the same fears held by the lay public about death, dying and hospices, to convey that the palliative care approach is applicable to many patients and is part of every doctors' role, whatever their specialty. Although facts and knowledge were thought to be important, coordinators were more concerned with attitudes and helping individuals with the transition from medical student to foundation doctor, providing an awareness of palliative medicine as a specialty and how to access it for their future patients.

Unique Genetic and Histological Signatures of Mouse Pericardial Adipose Tissue.

Obesity is a major risk factor for a plethora of metabolic disturbances including diabetes and cardiovascular disease. Accumulating evidence is showing that there is an adipose tissue depot-dependent relationship with obesity-induced metabolic dysfunction. While some adipose depots, such as subcutaneous fat, are generally metabolically innocuous, others such as visceral fat, are directly deleterious. A lesser known visceral adipose depot is the pericardial adipose tissue depot. We therefore set out to examine its transcriptional and morphological signature under chow and high-fat fed conditions, in comparison with other adipose depots, using a mouse model. Our results revealed that under chow conditions pericardial adipose tissue has uncoupling-protein 1 gene expression levels which are significantly higher than classical subcutaneous and visceral adipose depots. We also observed that under high-fat diet conditions, the pericardial adipose depot exhibits greatly upregulated transcript levels of inflammatory cytokines. Our results collectively indicate, for the first time, that the pericardial adipose tissue possesses a unique transcriptional and histological signature which has features of both a beige (brown fat-like) but also pro-inflammatory depot, such as visceral fat. This unique profile may be involved in metabolic dysfunction associated with obesity.

Mode of induction of platelet-derived extracellular vesicles is a critical determinant of their phenotype and function.

Platelet-derived extracellular vesicles (PDEVs) are the most abundant amongst all types of EVs in the circulation. However, the mechanisms leading to PDEVs release, their role in coagulation and phenotypic composition are poorly understood. PDEVs from washed platelets were generated using different stimuli and were characterised using nanoparticle tracking analysis. Procoagulant properties were evaluated by fluorescence flow cytometry and calibrated automated thrombography. EVs from plasma were isolated and concentrated using a novel protocol involving a combination of size exclusion chromatography and differential centrifugation, which produces pure and concentrated EVs. Agonist stimulation enhanced PDEV release, but did not alter the average size of EVs compared to those produced by unstimulated platelets. Agonist stimulation led to lower negatively-charged phospholipid externalization in PDEVs, which was reflected in the lower procoagulant activity compared to those generated without agonist stimulation. Circulating EVs did not have externalized negatively-charged phospholipids. None of the 4 types of EVs presented tissue factor. The mechanism by which PDEV formation is induced is a critical determinant of its phenotype and function. Importantly, we have developed methods to obtain clean, concentrated and functional EVs derived from platelet-free plasma and washed platelets, which can be used to provide novel insight into their biological functions.

Microtubules in the formation and development of the primary mesenchyme in Arbacia punctulata. II. An experimental analysis of their role in development and maintenance of cell shape.

TO EXPERIMENTALLY TEST THE SUGGESTION MADE IN THE PRECEDING PAPER THAT THE MICROTUBULES ARE INVOLVED IN CELL SHAPE DEVELOPMENT DURING THE FORMATION AND DIFFERENTIATION OF THE PRIMARY MESENCHYME, WE APPLIED TO THE EMBRYOS TWO TYPES OF AGENTS WHICH AFFECT CYTOPLASMIC MICROTUBULES: (a) colchicine and hydrostatic pressure, which cause the microtubules to disassemble, and (b) D(2)O, which tends to stabilize them. When the first type of agent is applied to sea urchin gastrulae, the development of the primary mesenchyme ceases, the microtubules disappear, and the cells tend to spherulate. With D(2)O development also ceases, but the tubules appear "frozen," and the cell asymmetries persist unaltered. These agents appear to block development by primarily interfering with the sequential disassembly and/or reassembly of microtubules into new patterns. The microtubules, therefore, appear to be influential in the development of cell form. On the other hand through a careful analysis of the action of these agents and others on both intra- and extracellular factors, we concluded that the microtubules do rather little for the maintenance of cell shape in differentiated tissues.

Microtubules in the formation and development of the primary mesenchyme in Arbacia punctulata. I. The distribution of microtubules.

Prior to gastrulation, the microtubules in the presumptive primary mesenchyme cells appear to diverge from points (satellites) in close association with the basal body of the cilium; from here most of the microtubules extend basally down the lateral margins of the cell. As these cells begin their migration into the blastocoel, they lose their cilia and adopt a spherical form. At the center of these newly formed mesenchyme cells is a centriole on which the microtubules directly converge and from which they radiate in all directions. Later these same cells develop slender pseudopodia containing large numbers of microtubules; the pseudopodia come into contact and fuse to form a "cable" of cytoplasm. Microtubules are now distributed parallel to the long axis of the cable and parallel to the stalks which connect the cell bodies of the mesenchyme cells to the cable. Microtubules are no longer connected to the centrioles in the cell bodies. On the basis of these observations we suggest that microtubules are a morphological expression of a framework which opeartes to shape cells. Since at each stage in the developmental sequence microtubules appear to originate (or insert) on different sites in the cytoplasm, the possibility is discussed that these sites may ultimately control the distribution of the microtubules and thus the developmental sequence of form changes.

Diagnosing dying in the acute hospital setting--are we too late?

Studies have shown that end-of-life care within the UK hospital setting is variable, and care pathways are now being advocated in the UK. This report presents results from an audit revealing that it is possible to anticipate a large proportion of deaths within an acute setting, but this is generally achieved very close to the end of life. Forty-nine per cent of patients were recognised as dying 24 hours or less before death, 17% between 24 and 36 hours before death, 21% between 36 and 72 hours before death, and 13% greater than 72 hours before death. It discusses the challenges around making the 'diagnosis of dying' and highlights that if clinicians do not feel confident in performing such a diagnosis, then patients cannot benefit from end-of-life care pathways. Instead of asking healthcare professionals to make accurate prognoses or diagnose dying, an environment needs to be created where teams feel comfortable in actively managing patients (appropriately) alongside considering their symptom control and planning for possible end-of-life care.

Future innovations in anti-platelet therapies.

Platelets have long been recognized to be of central importance in haemostasis, but their participation in pathological conditions such as thrombosis, atherosclerosis and inflammation is now also well established. The platelet has therefore become a key target in therapies to combat cardiovascular disease. Anti-platelet therapies are used widely, but current approaches lack efficacy in a proportion of patients, and are associated with side effects including problem bleeding. In the last decade, substantial progress has been made in understanding the regulation of platelet function, including the characterization of new ligands, platelet-specific receptors and cell signalling pathways. It is anticipated this progress will impact positively on the future innovations towards more effective and safer anti-platelet agents. In this review, the mechanisms of platelet regulation and current anti-platelet therapies are introduced, and strong, and some more speculative, potential candidate target molecules for future anti-platelet drug development are discussed.

Pain assessment tools in palliative care: an urgent need for consensus.

At present, there is no universally accepted cancer pain assessment tool for use in palliative care (PC). The European Palliative Care Research Collaborative (EPCRC), therefore, aims to develop an international consensus-based computerised pain assessment tool. As part of this process, we have performed (1) a literature review on pain assessment tools for use in the PC and (2) an international expert survey to gain information on the relevant dimensions for pain assessment in PC. 230 publications were identified, only six met the inclusion criteria. Three further articles were identified through manual searching, totalling 11 different pain assessment tools. Nine tools were multidimensional. Pain intensity was assessed in seven, using various numerical/verbal rating scales (NRS/VRS); five tools focused on pain management. Three publications did not identify the rationale for the need to develop a new tool, and the selection procedure for items/dimensions was not described in six tools. Patient and/or professional expert groups were involved in the development of five tools and only two tools were extensively validated or cross-culturally tested. Thirty-two experts (71%) completed the expert survey and identified 'intensity', 'temporal pattern', 'relief/exacerbation', 'pain quality' and 'location' as the five most relevant dimensions. Most preferred assessment of 'pain intensity' was by NRS rather than VRS. Time windows extending 24 h were regarded as less relevant. Development of PC pain assessment tools seems to be a continuous process, which does not adhere to systematic guidelines, thus does not contribute to a universally accepted tool. No tool contained all relevant dimensions as defined by the experts. Many tools focused on particular dimensions, suggesting that specific research interests may drive the tool development process. Extensive literature reviews, expert and patient input and clinical studies are a needed approach in the development of a new consensus-based pain assessment tool.

Modeling thrombosis in silico: Frontiers, challenges, unresolved problems and milestones.

Hemostasis is a complex physiological mechanism that functions to maintain vascular integrity under any conditions. Its primary components are blood platelets and a coagulation network that interact to form the hemostatic plug, a combination of cell aggregate and gelatinous fibrin clot that stops bleeding upon vascular injury. Disorders of hemostasis result in bleeding or thrombosis, and are the major immediate cause of mortality and morbidity in the world. Regulation of hemostasis and thrombosis is immensely complex, as it depends on blood cell adhesion and mechanics, hydrodynamics and mass transport of various species, huge signal transduction networks in platelets, as well as spatiotemporal regulation of the blood coagulation network. Mathematical and computational modeling has been increasingly used to gain insight into this complexity over the last 30 years, but the limitations of the existing models remain profound. Here we review state-of-the-art-methods for computational modeling of thrombosis with the specific focus on the analysis of unresolved challenges. They include: a) fundamental issues related to physics of platelet aggregates and fibrin gels; b) computational challenges and limitations for solution of the models that combine cell adhesion, hydrodynamics and chemistry; c) biological mysteries and unknown parameters of processes; d) biophysical complexities of the spatiotemporal networks' regulation. Both relatively classical approaches and innovative computational techniques for their solution are considered; the subjects discussed with relation to thrombosis modeling include coarse-graining, continuum versus particle-based modeling, multiscale models, hybrid models, parameter estimation and others. Fundamental understanding gained from theoretical models are highlighted and a description of future prospects in the field and the nearest possible aims are given.

A humanized monoclonal antibody that inhibits platelet-surface ERp72 reveals a role for ERp72 in thrombosis.

Essentials ERp72 is a thiol isomerase enzyme. ERp72 levels increase at the platelet surface during platelet activation. We generated a humanized monoclonal antibody which blocks ERp72 enzyme activity (anti-ERp72). Anti-ERp72 inhibits platelet functional responses and thrombosis. SUMMARY: Background Within the endoplasmic reticulum, thiol isomerase enzymes modulate the formation and rearrangement of disulfide bonds in newly folded proteins entering the secretory pathway to ensure correct protein folding. In addition to their intracellular importance, thiol isomerases have been recently identified to be present on the surface of a number of cell types where they are important for cell function. Several thiol isomerases are known to be present on the resting platelet surface, including PDI, ERp5 and ERp57, and levels are increased following platelet activation. Inhibition of the catalytic activity of these enzymes results in diminished platelet function and thrombosis. Aim We previously determined that ERp72 is present at the resting platelet surface and levels increase upon platelet activation; however, its functional role on the cell surface was unclear. We aimed to investigate the role of ERp72 in platelet function and its role in thrombosis. Methods Using HuCAL technology, fully humanized Fc-null anti-ERp72 antibodies were generated. Eleven antibodies were screened for their ability to inhibit ERp72 activity and the most potent inhibitory antibody (anti-ERp72) selected for further testing in platelet functional assays. Results and conclusions Anti-ERp72 inhibited platelet aggregation, granule secretion, calcium mobilisation and integrin activation, revealing an important role for extracellular ERp72 in the regulation of platelet activation. Consistent with this, infusion of anti-ERp72 into mice protected against thrombosis.

The chaperone protein HSP47: a platelet collagen binding protein that contributes to thrombosis and hemostasis.

Essentials Heat shock protein 47 (HSP47), a collagen specific chaperone is present on the platelet surface. Collagen mediated platelet function was reduced following blockade or deletion of HSP47. GPVI receptor regulated signalling was reduced in HSP47 deficient platelets. Platelet HSP47 tethers to exposed collagen thus modulating thrombosis and hemostasis. SUMMARY: Objective Heat shock protein 47 (HSP47) is an intracellular chaperone protein that is vital for collagen biosynthesis in collagen secreting cells. This protein has also been shown to be present on the surface of platelets. Given the importance of collagen and its interactions with platelets in triggering hemostasis and thrombosis, in this study we sought to characterize the role of HSP47 in these cells. Methods and Results The deletion of HSP47 in mouse platelets or its inhibition in human platelets reduced their function in response to collagen and the GPVI agonist (CRP-XL), but responses to thrombin were unaltered. In the absence of functional HSP47, the interaction of collagen with platelets was reduced, and this was associated with reduced GPVI-collagen binding, signalling and platelet activation. Thrombus formation on collagen, under arterial flow conditions, was also decreased following the inhibition or deletion of HSP47, in the presence or absence of eptifibatide, consistent with a role for HSP47 in enhancing platelet adhesion to collagen. Platelet adhesion under flow to von Willebrand factor was unaltered following HSP47 inhibition. Laser-induced thrombosis in cremaster muscle arterioles was reduced and bleeding time was prolonged in HSP47-deficient mice or following inhibition of HSP47. Conclusions Our study demonstrates the presence of HSP47 on the platelet surface, where it interacts with collagen, stabilizes platelet adhesion and increases collagen-mediated signalling and therefore thrombus formation and hemostasis.

PPARγ agonists negatively regulate αIIbß3 integrin outside-in signaling and platelet function through up-regulation of protein kinase A activity.

Essentials peroxisome proliferator-activated receptor γ (PPARγ) agonists inhibit platelet function. PPARγ agonists negatively regulate outside-in signaling via integrin αIIbß3. PPARγ agonists disrupt the interaction of Gα13 with integrin ß3. This is attributed to an upregulation of protein kinase A activity. SUMMARY: Background Agonists for the peroxisome proliferator-activated receptor (PPARγ) have been shown to have inhibitory effects on platelet activity following stimulation by GPVI and GPCR agonists. Objectives Profound effects on thrombus formation led us to suspect a role for PPARγ agonists in the regulation of integrin αIIbß3 mediated signaling. Both GPVI and GPCR signaling pathways lead to αIIbß3 activation, and signaling through αIIbß3 plays a critical role in platelet function and normal hemostasis. Methods The effects of PPARγ agonists on the regulation of αIIbß3 outside-in signaling was determined by monitoring the ability of platelets to adhere and spread on fibrinogen and undergo clot retraction. Effects on signaling components downstream of αIIbß3 activation were also determined following adhesion to fibrinogen by Western blotting. Results Treatment of platelets with PPARγ agonists inhibited platelet adhesion and spreading on fibrinogen and diminished clot retraction. A reduction in phosphorylation of several components of αIIbß3 signaling, including the integrin ß3 subunit, Syk, PLCγ2, focal adhesion kinase (FAK) and Akt, was also observed as a result of reduced interaction of the integrin ß3 subunit with Gα13. Studies of VASP phosphorylation revealed that this was because of an increase in PKA activity following treatment with PPARγ receptor agonists. Conclusions This study provides further evidence for antiplatelet actions of PPARγ agonists, identifies a negative regulatory role for PPARγ agonists in the control of integrin αIIbß3 outside-in signaling, and provides a molecular basis by which the PPARγ agonists negatively regulate platelet activation and thrombus formation.

Platelet nitric oxide synthase is activated by tyrosine dephosphorylation: possible role for SHP-1 phosphatase.

BACKGROUND: Endothelial nitric oxide synthase (eNOS) activity in endothelial cells is regulated by post-translational phosphorylation of critical serine, threonine and tyrosine residues in response to a variety of stimuli. However, the post-translational regulation of eNOS in platelets is poorly defined. OBJECTIVES: We investigated the role of tyrosine phosphorylation in the regulation of platelet eNOS activity. METHODS: Tyrosine phosphorylation of eNOS and interaction with the tyrosine phosphatase SHP-1 were investigated by coimmunoprecipitation and immunoblotting. An in vitro immunoassay was used to determine eNOS activity together with the contribution of protein tyrosine phosphorylation. RESULTS: We found platelet eNOS was tyrosine phosphorylated under basal conditions. Thrombin induced a dose- and time-dependent increase in eNOS activity without altering overall level of tyrosine phosphorylation, although we did observe evidence of minor tyrosine dephosphorylation. In vitro tyrosine dephosphorylation of platelet eNOS using a recombinant protein tyrosine phosphatase enhanced thrombin-induced activity compared to thrombin alone, but had no effect on endothelial eNOS activity either at basal or after stimulation with bradykinin. Having shown that dephosphorylation could modulate platelet eNOS activity we examined the role of potential protein phosphatases important for platelet eNOS activity. We found SHP-1 protein tyrosine phosphatase, co-associated with platelet eNOS in resting platelets, but does not associate with eNOS in endothelial cells. Stimulation of platelets with thrombin increased SHP-1 association with eNOS, while inhibition of SHP-1 abolished the ability of thrombin to induce elevated eNOS activity. CONCLUSIONS: Our data suggest a novel role for tyrosine dephosphorylation in platelet eNOS activation, which may be mediated by SHP-1.

Loss of keratin expression in anaplastic carcinoma cells due to posttranscriptional down-regulation acting in trans.

Rat keratin K5 and vimentin complementary DNAs have been isolated, identified, and used to study keratin and vimentin expression as markers for cell differentiation. Isologous rat neoplastic epithelial cell lines used were based on a clonal benign epithelial line (A5P/B10) and a clonal anaplastic malignant derivative line (T952/F7). Stable cytoplasmic mRNA was detected for keratin but not vimentin in the benign cells. The anaplastic derivative cells expressed vimentin but showed a 1000-fold reduction in the keratin message, which nuclear run-on assays identified as being due to posttranscriptional down-regulation. An identical pattern of posttranscriptional down-regulation was found in independent malignant somatic cell hybrids of the benign and anaplastic cells. trans-acting regulatory mechanisms implicated in posttranscriptional (pretranslational) keratin down-regulation in these anaplastic malignant cells may play a role in the apparent loss of differentiation evident in tumor progression.

Platelet signaling: a complex interplay between inhibitory and activatory networks.

The role of platelets in hemostasis and thrombosis is dependent on a complex balance of activatory and inhibitory signaling pathways. Inhibitory signals released from the healthy vasculature suppress platelet activation in the absence of platelet receptor agonists. Activatory signals present at a site of injury initiate platelet activation and thrombus formation; subsequently, endogenous negative signaling regulators dampen activatory signals to control thrombus growth. Understanding the complex interplay between activatory and inhibitory signaling networks is an emerging challenge in the study of platelet biology, and necessitates a systematic approach to utilize experimental data effectively. In this review, we will explore the key points of platelet regulation and signaling that maintain platelets in a resting state, mediate activation to elicit thrombus formation, or provide negative feedback. Platelet signaling will be described in terms of key signaling molecules that are common to the pathways activated by platelet agonists and can be described as regulatory nodes for both positive and negative regulators.

The identification, purification and characterisation of an inhibitor of collagenase (20K) produced by neoplastic epithelial cells.

A rat carcinoma cell line (T2/H7) constitutively synthesised interstitial collagenase. When these cells were incubated with 12-O-tetradecanoylphorbol 13-acetate (TPA) they secreted an inhibitor of collagenase, which resulted in a net decrease of collagenolytic activity being detected in conditioned medium. Using reverse zymography, the Mr of the inhibitor was found to be 20,000 which suggests that it may be the rat homologue of inhibitor of metalloproteinase 2 (IMP2; TIMP-2), as it inhibited both the gelatinolytic and collagenolytic activities of rat collagenase. The inhibitor was separated from collagenase by filtration through a YM30 membrane. The inhibitor was purified further by sequential chromatography on heparin-Sepharose and Con A-Sepharose. It bound to heparin-Sepharose in 75 mM NaCl and was eluted with 300 mM NaCl. It did not bind to Con A-Sepharose, suggesting that it was a non-glycosylated molecule. The inhibitor was resistant to treatment with either trypsin, APMA or heat.

R-twist gene expression during rat palatogenesis.

Palatal clefting is often associated with premature fusion of cranial sutures in human craniosynostosis syndromes, many of which are characterised by mutations affecting the fibroblast growth factor receptor (FGFR) gene family. In palatal fusion, epithelio-mesenchymal transition (EMT) contributes to the dispersion of the midline epithelial seam. EMT has also been observed in neoplastic epithelial cells in relation to the acquisition of malignant characteristics where morphological changes are accompanied by rapid switching in the expression of fgfr2 from the epithelial type (kgfr) to the mesenchymal type (bek). The twist gene codes for a basic helix-loop-helix transcription factor putatively involved in regulation of transcription of fgfr2. Mutations in the TWIST gene have been described as being responsible for the Saethre-Chotzen syndrome, an autosomal dominant craniosynostosis associated with cleft palate as well as other disturbances of the facial skeleton. In this study we have analysed the distribution of twist transcripts during rat palatogenesis in vivo from 14.5 to 17.5 days post coitum by in situ hybridisation with digoxygenin-labelled ssDNA probes. twist transcripts were found to be concentrated in mesenchymal cells beneath the epithelium at the tip of the palatal shelves immediately prior to, and during fusion as well as in a localised epithelial area at the tip of the shelves prior to fusion, thereby implicating twist gene expression in the process of palatogenesis. This pattern of expression illuminates the disturbances of maxillary growth that occur in human craniosynostotic syndromes.

Rapid disappearance of the medial epithelial seam during palatal fusion occurs by multifocal breakdown that is preceded by expression of alpha smooth muscle actin in the epithelium.

Breakdown of the medial epithelial seam (MES) is essential to allow bridging of the mesenchyme during palatal fusion. Evidence exists for three mechanisms for this breakdown that are incompatible at the level of individual cells in the seam. To determine if breakdown of the seam was regionally restricted, 3-dimensional reconstructions were generated using volume rendering software from 1 micron serial sections in the sagittal plane of rat palates fixed during the process of fusion. The earliest break detected in electron micrographs was cell separation and in reconstructions was a discrete defect, with a rounded outline, nearer to the nasal than to the oral margin of the seam. Further breakdown produced a pattern of rounded defects along the nasal margin of the seam resulting in interconnected columns of cells preferentially attached to the oral epithelium. Computer generated slicing of reconstructed seams showed that groups of cells evident in cross-sections as islands at this stage of breakdown of the MES could be artifacts. Unequivocal islands of epithelial cells formed later in fusion had a rounded outline, an incomplete basal lamina and a halo of cells containing phagocytosed apoptotic debris. The pattern of breakdown indicated that the MES breaks down under tension. Laser confocal microscopy of sections and whole-mounts of palates demonstrated alpha-smooth muscle actin preferentially localized in the epithelial cells of the palatal shelves immediately before and during formation of the seam. Expression in epithelial cells of the isoform of actin normally restricted to smooth muscle cells engaged in tonic contraction supported an interpretation that the epithelial cells of the seam may be capable of generating tension during the palatal fusion event.