Repressor element 1 silencing transcription factor couples loss of pluripotency with neural induction and neural differentiation

Neural differentiation of embryonic stem cells (ESCs) requires coordinated repression of the pluripotency regulatory program and reciprocal activation of the neurogenic regulatory program. Upon neural induction, ESCs rapidly repress expression of pluripotency genes followed by staged activation of neural progenitor and differentiated neuronal and glial genes. The transcriptional factors that underlie maintenance of pluripotency are partially characterized whereas those underlying neural induction are much less explored, and the factors that coordinate these two developmental programs are completely unknown. One transcription factor, REST (repressor element 1 silencing transcription factor), has been linked with terminal differentiation of neural progenitors and more recently, and controversially, with control of pluripotency. Here, we show that in the absence of REST, coordination of pluripotency and neural induction is lost and there is a resultant delay in repression of pluripotency genes and a precocious activation of both neural progenitor and differentiated neuronal and glial genes. Furthermore, we show that REST is not required for production of radial glia-like progenitors but is required for their subsequent maintenance and differentiation into neurons, oligodendrocytes, and astrocytes. We propose that REST acts as a regulatory hub that coordinates timely repression of pluripotency with neural induction and neural differentiation.

Follistatin complexes Myostatin and antagonises Myostatin-mediated inhibition of myogenesis

Follistatin is known to antagonise the function of several members of the TGF-beta family of secreted signalling factors, including Myostatin, the most powerful inhibitor of muscle growth characterised to date. In this study, we compare the expression of Myostatin and Follistatin during chick development and show that they are expressed in the vicinity or in overlapping domains to suggest possible interaction during muscle development. We performed yeast and mammalian two-hybrid studies and show that Myostatin and Follistatin interact directly. We further show that single modules of the Follistatin protein cannot associate with Myostatin suggesting that the entire protein is required for the interaction. We analysed the interaction kinetics of the two proteins and found that Follistatin binds Myostatin with a high affinity of 5.84 x 10(-10) M. We next tested whether Follistatin suppresses Myostatin activity during muscle development. We confirmed our previous observation that treatment of chick limb buds with Myostatin results in a severe decrease in the expression of two key myogenic regulatory genes Pax-3 and MyoD. However, in the presence of Follistatin, the Myostatin-mediated inhibition of Pax-3 and MyoD expression is blocked. We additionally show that Myostatin inhibits terminal differentiation of muscle cells in high-density cell cultures of limb mesenchyme (micromass) and that Follistatin rescues muscle differentiation in a concentration-dependent manner. In summary, our data suggest that Follistatin antagonises Myostatin by direct protein interaction, which prevents Myostatin from executing its inhibitory effect on muscle development. (C) 2004 Elsevier Inc. All rights reserved.

Targeting the cell cycle machinery for the treatment of cardiovascular disease

Cardiovascular disease represents a major clinical problem affecting a significant proportion of the world's population and remains the main cause of death in the UK. The majority of therapies currently available for the treatment of cardiovascular disease do not cure the problem but merely treat the symptoms. Furthermore, many cardioactive drugs have serious side effects and have narrow therapeutic windows that can limit their usefulness in the clinic. Thus, the development of more selective and highly effective therapeutic strategies that could cure specific cardiovascular diseases would be of enormous benefit both to the patient and to those countries where healthcare systems are responsible for an increasing number of patients. In this review, we discuss the evidence that suggests that targeting the cell cycle machinery in cardiovascular cells provides a novel strategy for the treatment of certain cardiovascular diseases. Those cell cycle molecules that are important for regulating terminal differentiation of cardiac myocytes and whether they can be targeted to reinitiate cell division and myocardial repair will be discussed as will the molecules that control vascular smooth muscle cell (VSMC) and endothelial cell proliferation in disorders such as atherosclerosis and restenosis. The main approaches currently used to target the cell cycle machinery in cardiovascular disease have employed gene therapy techniques. We will overview the different methods and routes of gene delivery to the cardiovascular system and describe possible future drug therapies for these disorders. Although the majority of the published data comes from animal studies, there are several instances where potential therapies have moved into the clinical setting with promising results.

Nerve terminal GABAA receptors activate Ca2+/calmodulin-dependent signaling to inhibit voltage-gated Ca2+ influx and glutamate release

-Aminobutyric acid type A (GABAA) receptors, a family of Cl-permeable ion channels, mediate fast synaptic inhibition as postsynaptically enriched receptors for -aminobutyric acid at GABAergic synapses. Here we describe an alternative type of inhibition mediated byGABAA receptors present on neocortical glutamatergic nerve terminals and examine the underlying signaling mechanism(s). By monitoring the activity of the presynaptic CaM kinase II/synapsin I signaling pathway in isolated nerve terminals, we demonstrate that GABAA receptor activation correlated with an increase in basal intraterminal [Ca2]i. Interestingly, this activation of GABAA receptors resulted in a reduction of subsequent depolarization-evoked Ca2 influx, which thereby led to an inhibition of glutamate release. To investigate how the observed GABAA receptor-mediated modulation operates, we determined the sensitivity of this process to the Na-K-2Cl cotransporter 1 antagonist bumetanide, as well as substitution of Ca2 with Ba2, or Ca2/calmodulin inhibition by W7. All of these treatments abolished the modulation by GABAA receptors. Application of selective antagonists of voltage-gated Ca2 channels (VGCCs) revealed that the GABAA receptor-mediated modulation of glutamate release required the specific activity of L- and R-type VGCCs. Crucially, the inhibition of release by these receptors was abolished in terminals isolated from R-type VGCC knock-out mice. Together, our results indicate that a functional coupling between nerve terminal GABAA receptors and L- or R-type VGCCs is mediated by Ca2/calmodulin-dependent signaling. This mechanism provides a GABA-mediated control of glutamatergic synaptic activity by a direct inhibition of glutamate release.

The four major N- and C-terminal splice variants of the excitatory amino acid transporter GLT-1 form cell surface homomeric and heteromeric assemblies

The L-glutamate transporter GLT-1 is an abundant CNS membrane protein of the excitatory amino acid transporter (EAAT) family which controls extracellular L-glutamate levels and is important in limiting excitotoxic neuronal death. Using RT-PCR, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N- and C-termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected HEK-293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent L-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (V5, HA or FLAG) into the second extracellular domain of each isoform allowed co-immunoprecipitation and tr-FRET studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms are able to combine to form homomeric and heteromeric assemblies, each of which are expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.

Molecular genetic differentiation in earthworms inhabiting a heterogeneous Pb-polluted landscape

A Pb-mine site situated on acidic soil, but comprising of Ca-enriched islands around derelict buildings was used to study the spatial pattern of genetic diversity in Lumbricus rubellus. Two distinct genetic lineages ('A' and 'B'), differentiated at both the mitochondrial (mtDNA COII) and nuclear level (AFLPs) were revealed with a mean inter-lineage mtDNA sequence divergence of approximately 13%, indicative of a cryptic species complex. AFLP analysis indicates that lineage A individuals within one central 'ecological island' site are uniquely clustered, with little genetic overlap with lineage A individuals at the two peripheral sites. FTIR microspectroscopy of Pb-sequestering chloragocytes revealed different phosphate profiles in residents of adjacent acidic and calcareous islands. Bioinformatics found over-representation of Ca pathway genes in ESTPb libraries. Subsequent sequencing of a Ca-transport gene, SERCA, revealed mutations in the protein's cytosolic domain. We recommend the mandatory genotyping of all individuals prior to field-based ecotoxicological assays, particularly those using discriminating genomic technologies.

Differentiation status of limbal epithelial cells cultured on intact and denuded amniotic membrane before and after air-lifting

We have investigated differences in bovine limbal epithelial cell differentiation when expanded upon intact (amniotic epithelial cells and basement membrane remaining) and denuded human amniotic membrane, a commonly used substrate in ophthalmic surgery for corneal stem cell transplantation. Ex vivo expansion of the epithelial cells, in supplemented media, continued for 2 weeks followed by 1 week under ‘air-lifting’ conditions. Before and after air-lifting the differentiated (K3/K12 positive) and undifferentiated (K14 positive) cells were quantified by immunohistochemistry, Western blotting and quantitative PCR. Limbal epithelial cells expanded upon amniotic membrane formed 4-6 stratified layers, both on intact and denuded amniotic membrane. On denuded amniotic membrane the proportion of differentiated cells remained unaltered following airlifting. Within cells grown on intact amniotic membrane, however, the number of differentiated cells increased significantly following air-lifting. These results have important implications for both basic and clinical research. Firstly, they show that bovine limbal epithelia can be used as an alternative source of cells for basic research investigating ex vivo limbal stem cells expansion. Secondly, these findings serve as a warning to clinicians that the affect of amniotic membrane on transplantable cells is not fully understood; the use of intact or denuded amniotic membrane can produce different results in terms of the amount of differentiation, once cells are exposed to the air.

CD27 mediates interleukin-2-independent clonal expansion of the CD8+ T cell without effector differentiation

The clonal expansion of antigen-specific CD8+ T cells in response to microbial infections is essential for adaptive immunity. Although IL-2 has been considered to be primarily responsible for this process, quantitatively normal expansion occurs in the absence of IL-2 receptor signaling. Here, we show that ligating CD27 on CD8+ T cells that have been stimulated through the T cell receptor causes their expansion in the absence of IL-2 by mediating two distinct cellular processes: enhancing cell cycling and promoting cell survival by maintaining the expression of IL-7 receptor alpha. This pathway for clonal expansion of the CD8+ T cell is not associated with the development of a capacity either for production of IFN-gamma or for cytotoxic T lymphocyte function and, therefore, is uncoupled from differentiation. Furthermore, ligating CD27 increases the threshold concentration at which IL-2 induces IFN-gamma-producing capability by the CD8+ T cell, suggesting that CD27 signaling may suppress effector differentiation. Finally, CD8+ T cells that have been stimulated by the TCR/CD27 pathway maintain their capacity for subsequent expansion and effector differentiation in response to a viral challenge in vivo. Thus, the TCR/CD27 pathway enables the CD8+ T cell to replicate by a process of self-renewal, which may contribute to the continuous generation of new effector CD8+ T cells in persistent viral infections.

Glucosinolate-accumulating S-cells in Arabidopsis leaves and flower stalks undergo programmed cell death at early stages of differentiation

Plant secondary metabolites glucosinolates (GSL) have important functions in plant resistance to herbivores and pathogens. We identified all major GSL that are accumulated in S-cells in Arabidopsis by MALDI-TOF MS, and estimated by LC-MS that the total GSL concentration in these cells is above 130 mM. The precise locations of the S-cells outside phloem bundles in rosette and cauline leaves and in flower stalks were visualised using sulphur mapping by cryo-SEM/EDX. S-cells contain up to 40% of total sulphur in flower stalk tissues. S-cells in emerging flower stalks and developing leaf tissues show typical signs of Programmed Cell Death (PCD) or apoptosis, such as chromatin condensation in the nucleus and blebbing of the membranes. TUNEL staining for DNA double strand breaks confirmed PCD in S-cells in postmeristematic tissues in the flower stalk as well as in the leaf. Our results show that S-cells in postmeristematic tissues proceed to an extreme degree of metabolic specialisation besides PCD. Accumulation and maintenance of a high concentration of GSL in these cells are accompanied by degradation of a number of cell organelles. The substantial changes in the cell composition during S-cell differentiation indicate the importance of this particular GSL-based phloem defence system. The specific anatomy of the S-cells and ability to accumulate specialised secondary metabolites is similar to that of the non-articulated laticifer cells in latex plants and thus indicates a common evolutionary origin.

Variation in the ovarian reserve Is linked to alterations in intrafollicular estradiol production and ovarian biomarkers of follicular differentiation and oocyte quality in cattle

The mechanisms whereby the high variation in numbers of morphologically healthy oocytes and follicles in ovaries (ovarian reserve) may have an impact onovarian function, oocyte quality, and fertility are poorly understood. The objective was to determine whether previously validated biomarkers for follicular differentiation and function, as well as oocyte quality differed between cattle with low versus a high antral follicle count (AFC). Ovaries were removed (n = 5 per group) near the beginning of the nonovulatory follicular wave, before follicles could be identified via ultrasonography as being dominant, from heifers with high versus a low AFC. The F1, F2, and F3 follicles were dissected and diameters determined. Follicular fluid and thecal, granulosal, and cumulus cells and the oocyte were isolated and subjected to biomarker analyses. Although the size and numerous biomarkers of differentiation, such as mRNAs for the gonadotropin receptors, were similar, intrafollicular concentrations of estradiol and the abundance of mRNAs for CYP19A1 in granulosal cells and ESR1, ESR2, and CTSB in cumulus cells were greater, whereas mRNAs for AMH in granulosal cells and TBC1D1 in thecal cells were lower for animals with low versus a high AFC during follicle waves. Hence, variation in the ovarian reserve may have an impact on follicular function and oocyte quality via alterations in intrafollicular estradiol production and expression of key genes involved in follicle-stimulating hormone action (AMH) and estradiol (CYP19A1) production by granulosal cells, function and survival of thecal cells (TBC1D1), responsiveness of cumulus cells to estradiol (ESR1, ESR2), and cumulus cell determinants of oocyte quality (CTSB).

Origin and evolution of flavivirus 5'UTRs and panhandles: trans-terminal duplications?

Flavivirus replication is mediated by interactions between complementary ssRNA sequences of the 5'- and 3'-termini that form dsRNA cyclisation stems or panhandles, varying in length, sequence and specific location in the mosquito-borne, tick-borne, non-vectored and non-classified flaviviruses. In this manuscript we manually aligned the flavivirus 5'UTRs and adjacent capsid genes and revealed significantly more homology than has hitherto been identified. Analysis of the alignments revealed that the panhandles represent evolutionary remnants of a long cyclisation domain that probably emerged through duplication of one of the UTR termini.

Ribonucleocapsid formation of severe acute respiratory syndrome coronavirus through molecular action of the N-terminal domain of N protein

Conserved among all coronaviruses are four structural proteins: the matrix (M), small envelope (E), and spike (S) proteins that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in the lumen. The N-terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding, while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C terminus of the N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17 A (monoclinic) and at 1.85 A (cubic), respectively, resolved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the beta-sheet core. The functionally important positively charged beta-hairpin protrudes out of the core, is oriented similarly to that in the IBV N-NTD, and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggests a common mode of RNA recognition, but they probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs suggests that they use different modes of both RNA recognition and oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.

Nuclear magnetic resonance structure of the N-terminal domain of nonstructural protein 3 from the severe acute respiratory syndrome coronavirus

This paper describes the structure determination of nsp3a, the N-terminal domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 3. nsp3a exhibits a ubiquitin-like globular fold of residues 1 to 112 and a flexibly extended glutamic acid-rich domain of residues 113 to 183. In addition to the four beta-strands and two alpha-helices that are common to ubiquitin-like folds, the globular domain of nsp3a contains two short helices representing a feature that has not previously been observed in these proteins. Nuclear magnetic resonance chemical shift perturbations showed that these unique structural elements are involved in interactions with single-stranded RNA. Structural similarities with proteins involved in various cell-signaling pathways indicate possible roles of nsp3a in viral infection and persistence.