Transmembrane TNF-α: structure, function and interaction with anti-TNF agents

Transmembrane TNF-α, a precursor of the soluble form of TNF-α, is expressed on activated macrophages and lymphocytes as well as other cell types. After processing by TNF-α-converting enzyme (TACE), the soluble form of TNF-α is cleaved from transmembrane TNF-α and mediates its biological activities through binding to Types 1 and 2 TNF receptors (TNF-R1 and -R2) of remote tissues. Accumulating evidence suggests that not only soluble TNF-α, but also transmembrane TNF-α is involved in the inflammatory response. Transmembrane TNF-α acts as a bipolar molecule that transmits signals both as a ligand and as a receptor in a cell-to-cell contact fashion. Transmembrane TNF-α on TNF-α-producing cells binds to TNF-R1 and -R2, and transmits signals to the target cells as a ligand, whereas transmembrane TNF-α also acts as a receptor that transmits outside-to-inside (reverse) signals back to the cells after binding to its native receptors. Anti-TNF agents infliximab, adalimumab and etanercept bind to and neutralize soluble TNF-α, but exert different effects on transmembrane TNF-α-expressing cells (TNF-α-producing cells). In the clinical settings, these three anti-TNF agents are equally effective for RA, but etanercept is not effective for granulomatous diseases. Moreover, infliximab induces granulomatous infections more frequently than etanercept. Considering the important role of transmembrane TNF-α in granulomatous inflammation, reviewing the biology of transmembrane TNF-α and its interaction with anti-TNF agents will contribute to understanding the bases of differential clinical efficacy of these promising treatment modalities

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Development of a capillary electrophoretic method for the separation and detection of resin acids

A method for the separation and detection of standard resin acids (RAs), commonly found in pulp mill effluent and known to bioaccumulate in fish bile, was optimized using cyclodextrin modified electrokinetic capillary electrophoresis (CD-EKC) with ultra violet (UV) and laser induced fluorescence (LIF) detection. Optimal separation conditions were found with RA standards using UV detection at 214 nm, with a 72 mM sodium borate buffer pH 9.25, containing 35 mM beta-cyclodextrin sulfobutyl ether (SPCD), 15 mM of methyl-beta-cyclodextrin (MECD) and a 37 cm capillary with an internal diameter of 50pm. This resulted in a 12-min separation and the identification of 9 peaks, with a LOD of 10 ppm. To enable increased sensitivity, RAs were derivatized using the fluorescent label 4-BrMMc. A method for extracting resin acids from spiked fish bile and pulp mill effluent was developed, the extracted samples were derivatized, separated and identified using CD-EKC with LIF detection. The method of extraction and derivatization using CD-EKC was applied to biological samples of contaminated effluent and fish bile. (Abstract shortened by UMI.

Derivatization of resin acids with a fluorescent label for cyclodextrin-modified electrophoretic separation

NRC publication: Ye

Separation of resin acids using cyclodextrin modified capillary electrophoresis

A cyclodextrin-modified capillary electrophoretic method has been developed for the analysis of eleven common resin acids using a pH 4.5, 20 mM sodium acetate buffer containing 10% acetonitrile, 20 mM methyl-\u3b2-cyclodextrin (MECD) and 30 mM sulfobutylether-\u3b2-cyclodextrin (SBCD) as buffer modifiers. At pH below their pKa (< 5.7\u20146.4) the resin acids were virtually unionized and insoluble; however, they formed water-soluble inclusion complexes with MECD (20 mM) or SBCD (30 mM) even at pH 4.5. The analytes were separated in 25 min and, with the exception of two pairs, 12- or 14-chlorodehydroabietic/12,14-dichlorodehydroabietic acid and dehydroabietic/palustric acid, the remaining resin acids were baseline-separated. Analysis time was significantly shortened (< 12 min) at pH 9.25 using 30 mM SBCD and 20 mM MECD in 20 mM sodium borate. Resin acids were baseline-separated with the exception of two pairs, pimaric/ sandaracopimaric acid and 12- or 14-chlorodehydroabietic/abietic acid. The addition of 7.5% methanol to the running buffer resolved the abietic acid peak. Both HPLC and micellar capillary electrokinetic chromatography using 20 mM deoxycholic acid, 10% acetonitrile in 20 mM sodium borate, pH 9.25, failed to resolve the resin acids. The simple capillary electrophoretic method developed would be useful for the rapid separation and characterization of several important resin acids in pulp mill effluents and other contaminated samples.NRC publication: Ye

Transcription Profiling of Cyclic AMP Signaling in Candida albicans

We used transcription profiling in Candida albicans to investigate cellular regulation involving cAMP. We found that many genes require the adenylyl cyclase Cdc35p for proper expression. These include genes encoding ribosomal subunit proteins and RNA polymerase subunit proteins, suggesting that growth could be controlled in part by cAMP-mediated modulation of gene expression. Other genes influenced by loss of adenylyl cyclase are involved in metabolism, the cell wall, and stress response and include a group of genes of unknown function that are unique to C. albicans. The profiles generated by loss of the adenylyl cyclase regulator Ras1p and a downstream effector Efg1p were also examined. The loss of Ras1p function disturbs the expression of a subset of the genes regulated by adenylyl cyclase, suggesting both that the primary role of Ras1p in transcriptional regulation involves its influence on the function of Cdc35p and that there are Ras1p independent roles for Cdc35p. The transcription factor Efg1p is also needed for the expression of many genes; however, these genes are distinct from those modulated by Cdc35p with the exception of a class of hyphal-specific genes. Therefore transcription profiling establishes that cAMP plays a key role in the overall regulation of gene expression in C. albicans, and enhances our detailed understanding of the circuitry controlling this regulation

The zinc cluster transcription factor Tac1p regulates PDR16 expression in Candida albicans.

The Candida albicans PDR16 gene, encoding a putative phosphatidylinositol transfer protein, is co-induced with the multidrug transporter genes CDR1 and CDR2 in azole-resistant (A(R)) clinical isolates and upon fluphenazine exposure of azole-susceptible (A(S)) cells, suggesting that it is regulated by Tac1p, the transcriptional activator of CDR genes. Deleting TAC1 in an A(R) isolate (5674) overexpressing PDR16, CDR1 and CDR2 decreased the expression of the three genes and fluconazole resistance to levels similar to those detected in the matched A(S) isolate (5457), demonstrating that Tac1p is responsible for PDR16 upregulation in that strain. Deleting TAC1 in the A(S) strain SC5314 abolished CDR2 induction by fluphenazine and decreased that of PDR16 and CDR1, uncovering the participation of an additional factor in the regulation of PDR16 and CDR1 expression. Sequencing of the TAC1 alleles identified one homozygous mutation in strain 5674, an Asn to Asp substitution at position 972 in the C-terminus of Tac1p. Introduction of the Asp(972) allele in a tac1Delta/Delta mutant caused high levels of fluconazole resistance and TAC1, PDR16, CDR1 and CDR2 constitutive induction. These results demonstrate that: (i) Tac1p controls PDR16 expression; (ii) Asn(972) to Asp(972) is a gain-of-function mutation; and (iii) Tac1p is positively autoregulated, directly or indirectly.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

High-affinity transporters for NAD+ precursors in Candida glabrata are regulated by Hst1 and induced in response to niacin limitation

The yeast Candida glabrata is an opportunistic pathogen of humans. C. glabrata is a NAD+ auxotroph, and its growth depends on the availability of niacin (environmental vitamin precursors of NAD+). We have previously shown that a virulence-associated adhesin, encoded by EPA6, is transcriptionally induced in response to niacin limitation. Here we used transcript profiling to characterize the transcriptional response to niacin limitation and the roles of the sirtuins Hst1, Hst2, and Sir2 in mediating this response. The majority of genes transcriptionally induced by niacin limitation are regulated by Hst1, suggesting that it is the primary sensor of niacin limitation in C. glabrata. We show that three highly induced genes, TNA1, TNR1, and TNR2, encode transporters which are necessary and sufficient for high-affinity uptake of NAD+ precursors. Strikingly, if a tna1 tnr1 tnr2 mutant is starved for niacin, it exhibits an extended lag phase, suggesting a central role for the transporters in restoring NAD+ homeostasis after niacin limitation. Lastly, we had previously shown that the adhesin encoded by EPA6 is induced during experimental urinary tract infection (UTI); we show here that EPA6 transcriptional induction during UTI is strongly enhanced in the tna1 tnr1 tnr2 mutant strain, implicating the transporters in the growth of C. glabrata during infection.Peer reviewed: YesNRC publication: Ye

Examining the lived experience of bullying: a review of the literature from an Australian perspective

Bullying in schools is a significant and continuing issue in education. This is despite widespread attention within the professional education community and beyond, into the wider public arena. In this paper, we review the existing literature on bullying in schools, with a particular focus on the Australian secondary school context, to develop a position that questions the bully/victim binary pervading public discourse and educational research. In doing this, we identify common themes within the literature including: definitions of bullying; responses and interventions to bullying; discourses of bullying in schools; and the role of stakeholders involved in managing and responding to bullying incidents. Based on this review, we argue that much of the literature approaches the topic from an individual and psychological point of view, and there are multiple problems related to both methodology and representation. There appears to be an absence of research about the broader social contexts and processes in which bullying occurs, while there is a strong argument for its importance. From this basis, we briefly speculate on alternative approaches that potentially address such concerns and allow for new approaches to a continuing problem, in Australia and internationally