The deposition of metal nanoparticles on carbon surfaces: the role of specific functional groups.

The enormous complexity of a typical heterogeneous catalyst makes understanding the development and properties of any active nanoparticles present extremely challenging. In the case of carbon based catalysts that difficulty is compounded by the variability of the carbon powders used. We have previously developed a strategy that addresses these problems by mimicking the catalyst preparation conditions very closely but using highly ordered pyrolytic graphite crystals (HOPG) as a model surface. This enables us to examine the effects of specific functional groups on nanoparticle formation. We report here an extension of our work characterising functional groups on the HOPG surface, using XPS and AFM to explore the deposition of gold from aqueous solution onto HOPG surfaces treated in a variety of ways to alter the surface functionality. The structure and oxidation state of the resulting nanoparticles depend critically on the nature of the functional groups present and offers some insight into the development of catalysts based on these materials. Hydroxyls are identified as key functional species, reducing gold ions to their metallic state whilst being oxidised themselves to carbonyls. Carbonyls meanwhile promote the nucleation of Au3+, creating a network of islands at the HOPG surface. The results have relevance not only to catalysts using activated carbons but also the new generation of materials based on graphene and carbon nanotubes.

Mutations in TGIF cause holoprosencephaly and link NODAL signalling to human neural axis determination.

Holoprosencephaly (HPE) is the most common structural defect of the developing forebrain in humans (1 in 250 conceptuses, 1 in 16,000 live-born infants). HPE is aetiologically heterogeneous, with both environmental and genetic causes. So far, three human HPE genes are known: SHH at chromosome region 7q36 (ref. 6); ZIC2 at 13q32 (ref. 7); and SIX3 at 2p21 (ref. 8). In animal models, genes in the Nodal signalling pathway, such as those mutated in the zebrafish mutants cyclops (refs 9,10), squint (ref. 11) and one-eyed pinhead (oep; ref. 12), cause HPE. Mice heterozygous for null alleles of both Nodal and Smad2 have cyclopia. Here we describe the involvement of the TG-interacting factor (TGIF), a homeodomain protein, in human HPE. We mapped TGIF to the HPE minimal critical region in 18p11.3. Heterozygous mutations in individuals with HPE affect the transcriptional repression domain of TGIF, the DNA-binding domain or the domain that interacts with SMAD2. (The latter is an effector in the signalling pathway of the neural axis developmental factor NODAL, a member of the transforming growth factor-beta (TGF-beta) family.) Several of these mutations cause a loss of TGIF function. Thus, TGIF links the NODAL signalling pathway to the bifurcation of the human forebrain and the establishment of ventral midline structures.

Cooperative binding of Ets-1 and core binding factor to DNA.

Two phorbol ester-inducible elements (beta E2 and beta E3) within the human T-cell receptor beta gene enhancer each contain consensus binding sites for the Ets and core binding factor (CBF) transcription factor families. Recombinant Ets-1 and purified CBF bound individually to beta E2 and beta E3, in which the Ets and core sites are directly adjacent. In this report, we show that CBF and Ets-1 bind together to beta E2 and beta E3 and that Ets-1-CBF-DNA complexes are favored over the binding of either protein alone to beta E2. Formation of Ets-1-CBF-DNA complexes increased the affinity of Ets-1-DNA interactions and decreased the rate of dissociation of CBF from DNA. Ets-1-CBF-DNA complexes were not observed when either the Ets or core site was mutated. The spatial requirements for the cooperative interaction of Ets-1 and CBF were analyzed by oligonucleotide mutagenesis and binding site selection experiments. Core and Ets sites were coselected, and there appeared to be little constraint on the relative orientation and spacing of the two sites. These results demonstrate that CBF and Ets-1 form a high-affinity DNA-binding complex when both of their cognate sites are present and that the relative spacing and orientation of the two sites are unimportant. Ets and core sites are found in several T-cell-specific enhancers, suggesting that this interaction is of general importance in T-cell-specific transcription.

Smad transcriptional corepressors in TGF beta family signaling.

The known Smad transcriptional repressors appear to play multiple roles in modulating TGF beta-activated transcriptional responses. As detailed in Fig. 4, in the [figure: see text] absence of TGF beta signals, Ski/Sno prevent the activation of transcription by Smad proteins that find their way to the nucleus. Following TGF beta stimulation, the interaction with Ski/Sno is lost and these proteins are degraded. The free, activated Smad complex then enters the nucleus, where it can form two different kinds of transcriptional complexes: one involving interactions with general transcriptional coactivators, resulting in transcriptional activation, and the alternate complex, in which coactivators are displaced by a complex of corepressors recruited via a protein such as TGIF. The relative levels of these two complexes formed appear to be determined by the levels of available Smad coactivators and corepressors present within the cell. Once Smad transcriptional complexes have been formed, they can be further modulated by corepressors in at least two ways. TGF beta itself appears to upregulate SnoN expression, perhaps resulting in negative feedback on the activating Smad complexes. The balance between coactivators and corepressors within the cell can also be altered by other signaling inputs, and it appears that the stabilization of TGIF in response to activation of the MAP kinase pathway is able to shift the balance towards transcriptional repression. The scheme of action of Smad corepressors, represented in Fig. 4, is based on the initial analyses of these factors, and the challenge for the future is to more fully understand the precise physiological roles of Smad corepressors. Determining the roles they play in modulating responses to TGF beta family ligands during development, together with an analysis of the contributions of mutations that affect Smad corepressor function to genetic diseases such as HPE and to cancer will also be of great interest. Additionally, a better understanding of the events within the nucleus following BMP signaling may reveal the presence not only of more BMP-specific Smad recruiters, but also of BMP Smad-specific corepressors.

Regulation of human T cell receptor beta gene expression by Ets-1.

Expression of the human TcR beta gene is controlled by an enhancer located 6kb 3' to the C beta 2 gene segment. The activity of this enhancer has been shown to be inducible with phorbol esters. Within the enhancer the beta E2 element is responsible for the major part of the inducibility, multimerised beta E2 alone is also highly phorbol ester inducible. The beta E2 element contains a consensus ets-binding site as well as a core motif, and we have shown that the beta E2 ets site binds both Ets-1 and Ets-2 in vitro and that purified core binding factor (CBF) can bind the core site present in beta E2. Mutations which specifically disrupt Ets-1 and Ets-2 binding abolish inducibility as well as reducing activity, whereas mutants which cannot bind CBF have only reduced basal activity. In Jurkat, which has a high level of endogenous Ets-1, multimerized beta E2 was inactive unless treated with PMA. However when transfected into cells with no detectable Ets-1 the beta E2 multimer was highly active in the absence of PMA. Co-transfection of an Ets-1 expression construct with the full enhancer into Jurkat cells led to a repression of enhancer activity, suggesting a repressive role for Ets-1. Co-transfection of Ets-1 was also able to repress strongly the activity of the beta E2 multimer. Repression of activity from both the full enhancer construct and the beta E2 multimer was most dramatic in the presence of PMA, suggesting that Ets-1 could block TcR beta activation. The Ets-1 expression construct used transactivated the HTLV-1 LTR which has also been shown to bind Ets-1. The repression of beta E2 activity by Ets-1 appears therefore to be specific. In conclusion, the combination of ets and core sites in beta E2 constitutes a novel inducible element, which is specifically transrepressed by Ets-1.

Differential induction of the NF-AT complex during restimulation and the induction of T-cell anergy.

Stimulation of human CD4+ T-cell clones through the T-cell receptor (TcR) by high doses of specific peptide results in the induction of a long-lived state of nonresponsiveness that has been called anergy. During the induction of anergy, T cells are phenotypically similar to cells responding to an immunogenic stimulus. The amount of TcR at the cell surface is downmodulated, whereas the CD2 and CD25 receptors are increased. When restimulated, however, anergic T cells fail to up-regulate transcription of the IL-2 gene and in consequence do not produce IL-2. In this study, we have compared the ability of various transcription factors to bind to their appropriate site on DNA. Factors were isolated from the nuclei of T cells that were in the induction phase of anergy or were undergoing activation. The pattern of binding activity in restimulated T cells is consistent with the pattern that has previously been shown to regulate T-cell-specific expression of the IL-2 and the beta chain of the TcR genes. The measured binding to a TCF-1 site is the same in the nuclei of resting, activated, and anergized cells. The inducible factors NK-kappa B, beta E2, CD28RC, and AP-1 are not expressed in resting cells and are twofold lower in anergized as compared with activated cells. In contrast, anergic T cells express approximately eightfold lower amounts of NF-AT, a member of the class of inducible factors that regulates IL-2 gene transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of Akt signaling in prostate induces a TGFß-mediated restraint on cancer progression and metastasis.

Mutations in the PTEN tumor suppressor gene are found in a high proportion of human prostate cancers, and in mice, Pten deletion induces high-grade prostate intraepithelial neoplasia (HGPIN). However, progression from HGPIN to invasive cancer occurs slowly, suggesting that tumorigenesis is subject to restraint. We show that Pten deletion, or constitutive activation of the downstream kinase AKT, activates the transforming growth factor (TGF)ß pathway in prostate epithelial cells. TGFß signaling is known to have a tumor suppressive role in many cancer types, and reduced expression of TGFß receptors correlates with advanced human prostate cancer. We demonstrate that in combination either with loss of Pten or expression of constitutively active AKT1, inactivation of TGFß signaling by deletion of the TGFß type II receptor gene relieves a restraint on tumorigenesis. This results in rapid progession to lethal prostate cancer, including metastasis to lymph node and lung. In prostate epithelium, inactivation of TGFß signaling alone is insufficient to initiate tumorigenesis, but greatly accelerates cancer progression. The activation of TGFß signaling by Pten loss or AKT activation suggests that the same signaling events that have key roles in tumor initiation also induce the activity of a pathway that restrains disease progression.

Pc2 and SUMOylation.

Polycomb proteins are key regulators of transcription in metazoan organisms. Recent work has shed light on the nature of the polycomb protein complexes in flies and mammalian cells. Multiple enzymatic activities have been shown to associate with polycomb complexes, including histone methyltransferase, histone deacetylase and ubiquitination activities, which are primarily directed towards the modification of chromatin structure. In addition to these chromatin-based functions, other potential roles for polycomb proteins exist. Here, we present a comparison of vertebrate Pc2 (polycomb 2 protein) homologues, and review the known functions of the mammalian Pc2 focusing on its role as a SUMO (small ubiquitin-related modifier) E3 ligase. Pc2 is an E3 for several SUMO substrates, but still appears to have a more limited repertoire than other SUMO E3s, perhaps due to its association with polycomb complexes. One possibility is that Pc2 represents a relatively specialized polycomb protein, which has additional functions to those associated with other mammalian Pc (polycomb protein) paralogues.

A novel Rap1p-interacting factor, Rif2p, cooperates with Rif1p to regulate telomere length in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Rap1 protein binds with high affinity to sites within the poly(C(1-3)A) tracts at telomeres, where it plays a role in both telomere length regulation and the initiation of telomeric silencing. Rap1p initiates silencing at telomeres by interacting through its carboxy-terminal domain with Sir3p and Sir4p, both of which are required for repression. This same domain of Rap1p also negatively regulates telomere elongation, through an unknown mechanism. We have identified a new Rap1-interacting factor (Rif2p) that plays a role in telomere length regulation. Rif2p has considerable functional similarities with a Rap1p-interacting factor (Rif1p) identified previously. Mutations in RIF1 or RIF2 (unlike mutations in the silencing genes SIR3 and SIR4) result in moderate telomere elongation and improved telomeric silencing. However, deletion of both RIF1 and RIF2 in the same cell results in a dramatic increase in telomere length, similar to that seen with a carboxy-terminal truncation of Rap1p. In addition, overexpression of either RIF1 or RIF2 decreases telomere length, and co-overexpression of these proteins can reverse the telomere elongation effect of overexpression of the Rap1p carboxyl terminus. Finally, we show that Rif1p and Rif2p can interact with each other in vivo. These results suggest that telomere length regulation is mediated by a protein complex consisting of Rif1p and Rif2p, each of which has distinct regulatory functions. One role of Rap1p in telomere length regulation is to recruit these proteins to the telomeres.

Multimerization of Hsp42p, a novel heat shock protein of Saccharomyces cerevisiae, is dependent on a conserved carboxyl-terminal sequence.

Rap1p is a transcriptional regulator of Saccharomyces cerevisiae, which plays roles in both transcriptional activation and silencing. To identify proteins involved in Rap1p-dependent regulation of transcription, we used the two-hybrid system to screen for Rap1p-interacting proteins. Two of the clones isolated from this screen encode a truncated protein with homology to small heat shock proteins (HSPs). Here we present an analysis of this novel S. cerevisiae HSP, which we name Hsp42p. Expression of HSP42 is regulated by a range of stress conditions similar to S. cerevisiae HSP26, with which Hsp42p shares most homology. However, HSP42 expression is more sensitive to increased salt concentration and to starvation and, in contrast to HSP26 is expressed in unstressed cells. Hsp42p interacts with itself in the two-hybrid assay. This interaction is dependent on a hydrophobic region which is conserved among small HSPs. Using bacterially expressed Hsp42p fusion proteins. we demonstrate that this is a direct interaction. Fractionation of yeast protein extracts by size demonstrates that all of the Hsp42p in these extracts is present in complexes with a molecular mass of greater than 200 kDa, suggesting that Hsp42p exists in high molecular mass complexes.

The interaction of the carboxyl terminus-binding protein with the Smad corepressor TGIF is disrupted by a holoprosencephaly mutation in TGIF.

The homeodomain protein TGIF represses transcription in part by recruiting histone deacetylases. TGIF binds directly to DNA to repress transcription or interacts with TGF-beta-activated Smads, thereby repressing genes normally activated by TGF-beta. Loss of function mutations in TGIF result in holoprosencephaly (HPE) in humans. One HPE mutation in TGIF results in a single amino acid substitution in a conserved PLDLS motif within the amino-terminal repression domain. We demonstrate that TGIF interacts with the corepressor carboxyl terminus-binding protein (CtBP) via this motif. CtBP, which was first identified by its ability to bind the adenovirus E1A protein, interacts both with gene-specific transcriptional repressors and with a subset of polycomb proteins. Efficient repression of TGF-beta-activated gene responses by TGIF is dependent on interaction with CtBP, and we show that TGIF is able to recruit CtBP to a TGF-beta-activated Smad complex. Disruption of the PLDLS motif in TGIF abolishes the interaction of CtBP with TGIF and compromises the ability of TGIF to repress transcription. Thus, at least one HPE mutation in TGIF appears to prevent CtBP-dependent transcriptional repression by TGIF, suggesting an important developmental role for the recruitment of CtBP by TGIF.

A Smad transcriptional corepressor.

Following TGFbeta receptor-mediated phosphorylation and association with Smad4, Smad2 moves into the nucleus, binds to target promoters in association with DNA-binding cofactors, and recruits coactivators such as p300/CBP to activate transcription. We identified the homeodomain protein TGIF as a Smad2-binding protein and a repressor of transcription. A TGFbeta-activated Smad complex can recruit TGIF and histone deacetylases (HDACs) to a Smad target promoter, repressing transcription. Thus, upon entering the nucleus, a Smad2-Smad4 complex may interact with coactivators, forming a transcriptional activation complex, or with TGIF and HDACs, forming a transcriptional repressor complex. Formation of one of these two mutually exclusive complexes is determined by the relative levels of Smad corepressors and coactivators within the cell.

Rap1p and telomere length regulation in yeast.

Telomere length in the yeast Saccharomyces cerevisiae is under stringent genetic control such that a narrow length distribution of TG1-3 repeats is observed. Previous studies have shown that Rap1p, which binds to the double-stranded telomeric repeats, plays a role in regulating repeat length: point mutations in the Rap1p C-terminus often result in a higher average telomere length and deletion of this region causes extreme telomere elongation. We have investigated further the role of Rap1p in this process. Our results suggest that telomere length is regulated by a negative feedback mechanism that can sense the number of Rap1p molecules bound at the chromosome end. This length regulatory mechanism requires two other proteins, Rif1p and Rif2p, that interact with each other and with the Rap1p C-terminus. Although the same C-terminal domain of Rap1p is also involved in the initiation of telomere position effect (telomeric transcriptional silencing), this Rap1p function appears to be separate from, and indeed antagonistic to, its role in telomere length regulation.

Epidermal growth factor signaling via Ras controls the Smad transcriptional co-repressor TGIF.

Smad transcription factors mediate the actions of transforming growth factor-beta (TGF-beta) cytokines during development and tissue homeostasis. TGF-beta receptor-activated Smad2 regulates gene expression by associating with transcriptional co-activators or co-repressors. The Smad co-repressor TGIF competes with the co-activator p300 for Smad2 association, such that TGIF abundance helps determine the outcome of a TGF-beta response. Small alterations in the physiological levels of TGIF can have profound effects on human development, as shown by the devastating brain and craniofacial developmental defects in heterozygotes carrying a hypomorphic TGIF mutant allele. Here we show that TGIF levels modulate sensitivity to TGF-beta-mediated growth inhibition, that TGIF is a short-lived protein and that epidermal growth factor (EGF) signaling via the Ras-Mek pathway causes the phosphorylation of TGIF at two Erk MAP kinase sites, leading to TGIF stabilization and favoring the formation of Smad2-TGIF co-repressor complexes in response to TGF-beta. These results identify the first mechanism for regulating TGIF levels and suggest a potential link for Smad and Ras pathway convergence at the transcriptional level.

TGIF2 interacts with histone deacetylase 1 and represses transcription.

TG-interacting factor (TGIF) is a transcriptional repressor, which represses transcription by binding directly to DNA or interacts with transforming growth factor beta (TGF beta)-activated Smads, thereby repressing TGF beta-responsive gene expression. Mutation of TGIF in humans causes holoprosencephaly, a severe genetic disorder affecting craniofacial development. Searching human expressed sequence tag data bases revealed the presence of clones encoding a TGIF-related protein (TGIF2), which contains two regions of high sequence identity with TGIF. Here we show that, like TGIF, TGIF2 recruits histone deacetylase, but in contrast to TGIF, is unable to interact with the corepressor CtBP. TGIF2 and TGIF have very similar DNA-binding homeodomains, and TGIF2 represses transcription when bound to DNA via a TGIF binding site. TGIF2 interacts with TGF beta-activated Smads and represses TGF beta-responsive transcription. TGIF2 appears to be a context-independent transcriptional repressor, which can perform similar functions to TGIF and may play a role in processes, which, when disrupted by mutations in TGIF, cause holoprosencephaly.

A 3' transcriptional enhancer regulates tissue-specific expression of the human CD2 gene.

A strong lymphocyte-specific transcriptional enhancer was identified within a DNase I hypersensitive site at the 3' end of the human CD2 gene. Full activity, in a transient expression assay, was contained within a region of 550 bp (minimal enhancer). T cells which express CD2 could use the enhancer to activate transcription from the reporter gene chloramphenicol acetyltransferase in the context of a heterologous promoter. Lower levels of transcription were detected in non-CD2-expressing T cells and in B cells. In contrast, the enhancer did not function in the epithelial cell line HeLa or in Colo 320 HSR, a cell line of neuroendocrine origin. Low levels of enhancement were detectable from two core regions, which acted synergistically with other cis-acting sequences to generate the complete enhancer. DNase I footprinting studies identified six cis-acting sequences to which proteins bound. Five of these sequence motifs were novel; the sixth was a canonical cAMP response element. Topoisomerase II, and scaffold attachment region consensus sequences were also found within an A/T-rich area downstream of the minimal enhancer. Neither region was bound to the nuclear matrix. The CD2 enhancer is modular in structure, it is constructed of novel cis-acting sequences and it is a major component of the regulatory system that controls expression of the CD2 gene.