R. Steven Conlan

Toll-like receptor and antimicrobial peptide expression in the bovine endometrium

BackgroundThe endometrium is commonly infected with bacteria leading to severe disease of the uterus in cattle and humans. The endometrial epithelium is the first line of defence for this mucosal surface against bacteria and Toll-like receptors (TLRs) are a critical component of the innate immune system for detection of pathogen associated molecular patterns (PAMPs). Antimicrobial peptides, acute phase proteins and Mucin-1 (MUC-1) also provide non-specific defences against microbes on mucosal surfaces. The present study examined the expression of innate immune defences in the bovine endometrium and tested the hypothesis that endometrial epithelial cells express functional receptors of the TLR family and the non-specific effector molecules for defence against bacteria.MethodsBovine endometrial tissue and purified populations of primary epithelial and stromal cells were examined using RT-PCR for gene expression of TLRs, antimicrobial peptides and MUC-1. Functional responses were tested by evaluating the secretion of prostaglandin E2 and acute phase proteins when cells were treated with bacterial PAMPs such as bacterial lipopolysaccharide (LPS) and lipoproteins.ResultsThe endometrium expressed TLRs 1 to 10, whilst purified populations of epithelial cells expressed TLRs 1 to 7 and 9, and stromal cells expressed TLRs 1 to 4, 6, 7, 9 and 10. The TLRs appear to be functional as epithelial cells secreted prostaglandin E2 in response to bacterial PAMPs. In addition, the epithelial cells expressed antimicrobial peptides, such as Tracheal and Lingual Antimicrobial Peptides (TAP and LAP) and MUC-1, which were upregulated when the cells were treated with LPS. However, the epithelial cells did not express appreciable amounts of the acute phase proteins haptoglobin or serum amyloid A.ConclusionEpithelial cells have an essential role in the orchestration of innate immune defence of the bovine endometrium and are likely to be the key to prevention of endometrial infection with bacteria.

The transcription corepressor LEUNIG interacts with the histone deacetylase HDA19 and mediator components MED14 (SWP) and CDK8 (HEN3) to repress transcription

ABSTRACT Transcription corepressors are general regulators controlling the expression of genes involved in multiple signaling pathways and developmental programs. Repression is mediated through mechanisms including the stabilization of a repressive chromatin structure over control regions and regulation of Mediator function inhibiting RNA polymerase II activity. Using whole-genome arrays we show that the Arabidopsis thaliana corepressor LEUNIG, a member of the GroTLE transcription corepressor family, regulates the expression of multiple targets in vivo. LEUNIG has a role in the regulation of genes involved in a number of different physiological processes including disease resistance, DNA damage response, and cell signaling. We demonstrate that repression of in vivo LEUNIG targets is achieved through histone deacetylase (HDAC)-dependent and -independent mechanisms. HDAC-dependent mechanisms involve direct interaction with HDA19, a class 1 HDAC, whereas an HDAC-independent repression activity involves interactions with the putative Arabidopsis Mediator components AtMED14/SWP and AtCDK8/HEN3. We suggest that changes in chromatin structure coupled with regulation of Mediator function are likely to be utilized by LEUNIG in the repression of gene transcription.

The Tup1-Cyc8 protein complex can shift from a transcriptional co- repressor to a transcriptional co-activator

Cyc8(Ssn6)-Tup1, a general co-repressor complex, is recruited to promoter DNA via interactions with DNA-binding regulatory proteins and inhibits the transcription of many different yeast genes. Previous studies have established that repression function of the complex is performed by one subunit of the complex, the Tup1 protein, and requires specific components of the RNA polymerase II holoenzyme such as Sin4 and Rgr1. In this study we test the transcriptional activity of the Cyc8 subunit using a lexAoperator-containing reporter. We show that a LexA-Cyc8 hybrid stimulates transcription when expressed in atup1Δ, a sin4Δ, or a rgr1Δstrain, suggesting that transcriptional activation is an intrinsic property of the Cyc8-Tup1 co-repressor. In support of this notion we demonstrate that Cyc8-Tup1 has a dual function on CIT2, a gene encoding a citrate synthase that is expressed upon mitochondrial dysfunction. First, we show that Cyc8-Tup1 is tethered toCIT2 promoter by interacting with the activation domain of Rtg3, a bHLH/L-Zip DNA-binding transactivator of CIT2. Next we demonstrate that Cyc8-Tup1 activates CIT2 transcription in response to mitochondrial dysfunction, and this stimulatory effect is mediated by Cyc8. In contrast, basal (noninduced) expression of this gene is inhibited by Tup1. These findings establish a positive role for the Cyc8-Tup1 complex in transcription and support a model by which specific metabolic signals may convert the Cyc8-Tup1 transcriptional co-repressor to a co-activator of certain promoters.

ISOLATION AND CHARACTERISATION OF CDNA CLONES REPRESENTING THE GENES ENCODING THE MAJOR TUBER STORAGE PROTEIN (DIOSCORIN) OF YAM (DIOSCOREA CAYENENSIS LAM.)

AbstractcDNA clones encoding dioscorins, the major tuber storage proteins (Mr 32000) of yam (Dioscorea cayenensis) have been isolated. Two classes of clone (A and B, based on hybrid release translation product sizes and nucleotide sequence differences) which are 84.1% similar in their protein coding regions, were identified. The protein encoded by the open reading frame of the class A cDNA insert is of Mr 30015. The difference in observed and calculated molecular mass might be attributed to glycosylation. Nucleotide sequencing and in vitro transcription/translation suggest that the class A dioscorin proteins are synthesised with signal peptides of 18 amino acid residues which are cleaved from the mature peptide. The class A and class B proteins are 69.6% similar with respect to each other, but show no sequence identity with other plant proteins or with the major tuber storage proteins of potato (patatin) or sweet potato (sporamin). Storage protein gene expression was restricted to developing tubers and was not induced by growth conditions known to induce expression of tuber storage protein genes in other plant species. The codon usage of the dioscorin genes suggests that the Dioscoreaceae are more closely related to dicotyledonous than to monocotyledonous plants.

High-resolution imaging using a novel atomic force microscope and confocal laser scanning microscope hybrid instrument: essential sample preparation aspects

The recent data explosion in global gene expression profiling and proteomics has resulted in a need to determine the mechanistic role of biomarker signatures in pathogenicity. Consequently, elaborate technologies are required to assess increasingly smaller sub-cellular compartments and constituents. We describe the development, evaluation and application of an efficient sample preparation methodology to facilitate coupled atomic force microscopy and confocal laser scanning microscopy (AFM–CLSM), providing a novel means of concurrent high-resolution structural and fluorescence imaging. Due to their fragile nature and nanoscale dimensions, filopodia were selected as a model to develop the procedure that maximised fluorescence response, while maintaining epithelial cell ultra-structure. Fixation with ultra-pure methanol-free formaldehyde coupled to quantum dot nanocrystal labelling proved to be vital in achieving high quality AFM–CLSM images. We demonstrated for the first time that filopodia have a “quilted” surface structure. Additionally, high ultra-structural ridges on the apical cell surface resolved by AFM corresponded to punctate moesin clusters, representing direct visualisation of moesin linkages between transmembrane proteins and the cytoskeleton. The capacity of this novel multi-modal imaging technique to probe topography, molecular composition and biophysical properties of ultra-structural features therefore provides unique information that will significantly contribute to our understanding of cellular structure–function relationships.

In vitro growth factor-induced bio engineering of mature articular cartilage

Articular cartilage maturation is the postnatal development process that adapts joint surfaces to their site-specific biomechanical demands. Maturation involves gross morphological changes that occur through a process of synchronised growth and resorption of cartilage and generally ends at sexual maturity. The inability to induce maturation in biomaterial constructs designed for cartilage repair has been cited as a major cause for their failure in producing persistent cell-based repair of joint lesions. The combination of growth factors FGF2 and TGFβ1 induces accelerated articular cartilage maturation in vitro such that many molecular and morphological characteristics of tissue maturation are observable. We hypothesised that experimental growth factor-induced maturation of immature cartilage would result in a biophysical and biochemical composition consistent with a mature phenotype. Using native immature and mature cartilage as reference, we observed that growth factor-treated immature cartilages displayed increased nano-compressive stiffness, decreased surface adhesion, decreased water content, increased collagen content and smoother surfaces, correlating with a convergence to the mature cartilage phenotype. Furthermore, increased gene expression of surface structural protein collagen type I in growth factor-treated explants compared to reference cartilages demonstrates that they are still in the dynamic phase of the postnatal developmental transition. These data provide a basis for understanding the regulation of postnatal maturation of articular cartilage and the application of growth factor-induced maturation in vitro and in vivo in order to repair and regenerate cartilage defects.

Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator

Significance Mediator is a megadalton multisubunit molecular switchboard involved in gene regulation in eukaryotes and is structurally conserved between species. It bridges the general transcription machinery and function-specific DNA binding proteins. It plays a dynamic role in regulating a wide range of processes, involving, for example, thyroid and vitamin D receptors. The role of Mediator appears to be in the fine tuning of the activation and repression of gene expression in many organisms, yet the underlying mechanisms of how its own function is regulated remains to be unraveled. Here we demonstrate how Mediator autoregulates its own function by cross-talk between the tail module and the Cdk8 kinase module in an active process involving priming of the mediator component Med3 for ubiquitin-ligase (Grr1)–mediated degradation by Cdk8 phosphorylation. Mediator, an evolutionary conserved large multisubunit protein complex with a central role in regulating RNA polymerase II–transcribed genes, serves as a molecular switchboard at the interface between DNA binding transcription factors and the general transcription machinery. Mediator subunits include the Cdk8 module, which has both positive and negative effects on activator-dependent transcription through the activity of the cyclin-dependent kinase Cdk8, and the tail module, which is required for positive and negative regulation of transcription, correct preinitiation complex formation in basal and activated transcription, and Mediator recruitment. Currently, the molecular mechanisms governing Mediator function remain largely undefined. Here we demonstrate an autoregulatory mechanism used by Mediator to repress transcription through the activity of distinct components of different modules. We show that the function of the tail module component Med3, which is required for transcription activation, is suppressed by the kinase activity of the Cdk8 module. Med3 interacts with, and is phosphorylated by, Cdk8; site-specific phosphorylation triggers interaction with and degradation by the Grr1 ubiquitin ligase, thereby preventing transcription activation. This active repression mechanism involving Grr1-dependent ubiquitination of Med3 offers a rationale for the substoichiometric levels of the tail module that are found in purified Mediator and the corresponding increase in tail components seen in cdk8 mutants.

PAH-Domain-Specific Interactions of the Arabidopsis Transcription Coregulator SIN3-LIKE1 (SNL1) with Telomere-Binding Protein 1 and ALWAYS EARLY2 Myb-DNA Binding Factors

The eukaryotic SIN3 protein is the central component of the evolutionarily conserved multisubunit SIN3 complex that has roles in regulating gene expression and genome stability. Here we characterise the structure of the SIN3 protein in higher plants through the analysis of SNL1 (SIN3-LIKE1), SNL2, SNL3, SNL4, SNL5 and SNL6, a family of six SIN3 homologues in Arabidopsis thaliana. In an Arabidopsis-protoplast beta-glucuronidase reporter gene assay, as well as in a heterologous yeast repression assay, full-length SNL1 was shown to repress transcription in a histone-deacetylase-dependent manner, demonstrating the conserved nature of SIN3 function. Yeast two-hybrid screening identified a number of DNA binding proteins each containing a single Myb domain that included the Arabidopsis ALWAYS EARLY proteins AtALY2 and AtALY3, and two telomere binding proteins AtTBP1 and AtTRP2/TRFL1 as SNL1 partners, suggesting potential functions for SNL1 in development and telomere maintenance. The interaction with telomere-binding protein 1 was found to be mediated through the well-defined paired amphipathic helix domain PAH2. In contrast, the AtALY2 interaction was mediated through the PAH3 domain of SNL1, which is structurally distinct from PAH1 and PAH2, suggesting that evolution of this domain to a more novel structural motif has occurred. These findings support a diverse role of SNL1 in the regulation of transcription and genome stability.

Tumor necrosis factor-α and interferon-γ stimulate MUC16 (CA125) expression in breast, endometrial and ovarian cancers through NFκB

Transmembrane mucins (TMs) are restricted to the apical surface of normal epithelia. In cancer, TMs not only are over-expressed, but also lose polarized distribution. MUC16/CA125 is a high molecular weight TM carrying the CA125 epitope, a well-known molecular marker for human cancers. MUC16 mRNA and protein expression was mildly stimulated by low concentrations of TNFα (2.5 ng/ml) or IFNγ (20 IU/ml) when used alone; however, combined treatment with both cytokines resulted in a moderate (3-fold or less) to large (> 10-fold) stimulation of MUC16 mRNA and protein expression in a variety of cancer cell types indicating that this may be a general response. Human cancer tissue microarray analysis indicated that MUC16 expression directly correlates with TNFα and IFNγ staining intensities in certain cancers. We show that NFκB is an important mediator of cytokine stimulation of MUC16 since siRNA-mediated knockdown of NFκB/p65 greatly reduced cytokine responsiveness. Finally, we demonstrate that the 250 bp proximal promoter region of MUC16 contains an NFκB binding site that accounts for a large portion of the TNFα response. Developing methods to manipulate MUC16 expression could provide new approaches to treating cancers whose growth or metastasis is characterized by elevated levels of TMs, including MUC16.

Polyaniline-graphene based α-amylase biosensor with a linear dynamic range in excess of 6 orders of magnitude.

α-amylase is an established marker for diagnosis of pancreatic and salivary disease, and recent research has seen a substantial expansion of its use in therapeutic and diagnostic applications for infection, cancer and wound healing. The lack of bedside monitoring devices for α-amylase detection has hitherto restricted the clinical progress of such applications. We have developed a highly sensitive α-amylase immunosensor platform, produced via in situ electropolymerization of aniline onto a screen-printed graphene support (SPE). Covalently binding an α-amylase specific antibody to a polyaniline (PANI) layer and controlling device assembly using electrochemical impedance spectroscopy (EIS), we have achieved a highly linear response against α-amylase concentration. Each stage of the assembly was characterized using a suite of high-resolution topographical, chemical and mechanical techniques. Quantitative, highly sensitive detection was demonstrated using an artificially spiked human blood plasma samples. The device has a remarkably wide limit of quantification (0.025-1000IU/L) compared to α-amylase assays in current clinical use. With potential for simple scale up to volume manufacturing though standard semiconductor production techniques and subsequently clinical application, this biosensor will enable clinical benefit through early disease detection, and better informed administration of correct therapeutic dose of drugs used to treat α-amylase related diseases.