The rise and decline in Salmonella enterica serovar Enteritidis outbreaks attributed to egg-containing foods in the United States, 1973-2009.

Salmonella enterica causes an estimated 1 million domestically acquired foodborne illnesses annually. Salmonella enterica serovar Enteritidis (SE) is among the top three serovars of reported cases of Salmonella. We examined trends in SE foodborne outbreaks from 1973 to 2009 using Joinpoint and Poisson regression. The annual number of SE outbreaks increased sharply in the 1970s and 1980s but declined significantly after 1990. Over the study period, SE outbreaks were most frequently attributed to foods containing eggs. The average rate of SE outbreaks attributed to egg-containing foods reported by states began to decline significantly after 1990, and the proportion of SE outbreaks attributed to egg-containing foods began declining after 1997. Our results suggest that interventions initiated in the 1990s to decrease SE contamination of shell eggs may have been integral to preventing SE outbreaks.

Listen while you work? The attitude of healthcare professionals to music in the operating theatre.

Although the playing of music is commonplace in the operating theatre, there is nothing in the literature examining whether staff feel this is beneficial. Questionnaires were distributed amongst a random selection of staff in practice at a district general hospital: medical staff from a range of surgical specialities, anaesthetists, and all grades of perioperative staff (nurse/operating department practitioners/healthcare assistants) were encouraged to participate. There were 121 health professionals in total working in the operating theatres. The authors compared the responses to each question amongst the respondents, to check for the tendency to correlate. Out of the 52 health professionals who responded, 36 stated that music is played in their theatre either every day, or two to three times a week. Only five respondents felt that this was too often. Fifteen percent of medical staff were of the opinion that the nursing staff controlled the choice of music. Nursing staff were almost evenly split in thinking that nursing staff, surgical staff and the whole theatre team controlled the choice of music. The majority of both nursing and medical staff felt that they enjoyed their work more and performed better when music was played in theatre. The study concluded that the majority of theatre staff found listening to music while they work a positive experience. The potential for music to have a distracting or detrimental effect on a minority of individuals should always be considered.

c-Myc induces changes in higher order rDNA structure on stimulation of quiescent cells.

c-Myc is an oncogenic transcription factor capable of activating transcription by all three nuclear RNA polymerases, thus acting as a high-level coordinator of protein synthesis capacity and cell growth rate. c-Myc recruits RNA polymerase I-related transcription factors to the rDNA when quiescent cells are stimulated to re-enter the cell cycle. Using a model system of cell lines with variable c-Myc status, we show that on stimulation c-Myc rapidly induces gene loop structures in rDNA chromatin that juxtapose upstream and downstream rDNA sequences. c-Myc activation is both necessary and sufficient for this change in rDNA chromatin conformation. c-Myc activation induces association of TTF-1 with the rDNA, and c-Myc is physically associated with induced rDNA gene loops. The origins of two or more rDNA gene loops are closely juxtaposed, suggesting the possibility that c-Myc induces nucleolar chromatin hubs. Induction of rDNA gene loops may be an early step in the reprogramming of quiescent cells as they re-enter the growth cycle.

Return to work following varicose vein surgery: influence of type of operation, employment and social status.

OBJECTIVES: To determine factors which influence the time taken to return to work in patients undergoing varicose vein surgery. DESIGN: Prospective collection of data from patients at outpatient interview. SETTING: The Department of Vascular and Endovascular Surgery at a teaching hospital in the UK. PARTICIPANTS: Two hundred and fifteen consecutive employed or self-employed patients attending the outpatient clinic for review following varicose vein surgery. METHODS: Data was collected from patients in the outpatient clinic approximately 6 weeks following varicose vein surgery. Type of procedure, gender, occupation status, category of occupation, the incidence of complications and the time taken to return to work (RTW) was recorded. Statistics were performed using Kruskal-Wallis H, Mann-Whitney U and chi-squared analysis. RESULTS: Two hundred and fifteen patients were included, 77 (36%) men and 138 (64%) women. One hundred and ninety-two (89%) were employed and 23 (11%) self-employed. One hundred and fifty-three underwent primary saphenofemoral (SFJ) surgery, 10 bilateral procedures, 23 primary saphenopopliteal surgery (SPJ), 14 redo operations, five combined SFJ and SPJ, two mid thigh perforator ligation, six phlebectomies without groin or popliteal surgery and two bilateral surgery for recurrence. There was no relationship of gender or incidence of complications to RTW. There was a significant difference (p<0.0001) between employed (median RTW 4 weeks, interquartile range 2-5 weeks) and self-employed patients (median 2 weeks, interquartile range 1-4 weeks). Occupation category did show an overall significant difference (p<0.0001) on Kruskal-Wallis H-testing. Paired Mann-Whitney U-analysis showed that this difference was between occupation class I (median RTW 2 weeks, interquartile range 1-3 weeks) and IIIN (median 3.5 weeks, interquartile range 2-5 weeks), IIIM (median 5 weeks, interquartile range 2-5 weeks), IV (median 4 weeks, interquartile range 2-6 weeks) and V (median 4 weeks interquartile range 3-6 weeks), and between class II (median 3 weeks, interquartile range 2-4 weeks) and classes IIIM, IV and V. CONCLUSIONS: Employed patients and those involved in intensive manual labour are less likely to return to work early. There is no effect of gender or incidence of complications. On the basis of this study we would recommend that patients could return to work within 3 weeks of varicose veins surgery.

The N-terminal regions of estrogen receptor alpha and beta are unstructured in vitro and show different TBP binding properties.

The N-terminal regions of the estrogen receptor alpha (ER alpha-N) and beta (ER beta-N) were expressed and purified to homogeneity. Using NMR and circular dichroism spectroscopy, we conclude that both ER alpha-N and ER beta-N are unstructured in solution. The TATA box-binding protein (TBP) has been shown previously to interact with ER alpha-N in vitro and to potentiate ER-activated transcription. We used surface plasmon resonance and circular dichroism spectroscopy to confirm and further characterize the ER-N-TBP interaction. Our results show that the intrinsically unstructured ER alpha-N interacts with TBP, and suggest that structural changes are induced in ER alpha-N upon TBP interaction. Conformational changes upon target factor interaction have not previously been demonstrated for any N-terminal region of nuclear receptors. In addition, no binding of ER beta-N to TBP was detected. This difference in TBP binding could imply differential recruitment of target proteins by ER alpha-N and ER beta-N. The affinity of the ER alpha-N-TBP interaction was determined to be in the micromolar range (K(D) = 10(-6) to 10(-5) m). Our results support models of TBP as a target protein for the N-terminal activation domain of ER alpha. Further, our results suggest that target proteins can induce and/or stabilize ordered structure in N-terminal regions of nuclear receptors upon interaction.

How transcriptional activators bind target proteins.

The product of the proto-oncogene c-myc influences many cellular processes through the regulation of specific target genes. Through its transactivation domain (TAD), c-Myc protein interacts with several transcription factors, including TATA-binding protein (TBP). We present data that suggest that in contrast to some other transcriptional activators, an extended length of the c-Myc TAD is required for its binding to TBP. Our data also show that this interaction is a multistep process, in which a rapidly forming low affinity complex slowly converts to a more stable form. The initial complex formation results from ionic or polar interactions, whereas the slow conversion to a more stable form is hydrophobic in nature. Based on our results, we suggest two alternative models for activation domain/target protein interactions, which together provide a single universal paradigm for understanding activator-target factor interactions.

Recruitment of chromatin remodelling factors during gene activation via the glucocorticoid receptor N-terminal domain.

We have shown that yeast mutants with defects in the Ada adaptor proteins are defective in hormone-dependent gene activation by ectopically expressed human glucocorticoid receptor (GR). Others have shown that the Ada2 protein is required for physical interactions between some activation domains and TBP (TATA-binding protein), whereas the Gcn5 (Ada4) protein has a histone acetyltransferase (HAT) activity. Although all HAT enzymes are able to acetylate histone substrates, some also acetylate non-histone proteins. Taken together, these observations suggest that the Ada proteins have the ability to effect different steps in the process of gene activation. It has recently been shown that the Ada proteins are present in two distinct protein complexes, the Ada complex and a larger SAGA complex. Our recent work has focused on determining (1) which of the Ada-containing complexes mediates gene activation by GR, (2) whether the HAT activity encoded by GCN5 is required for GR-dependent gene activation, (3) whether the Ada proteins contribute to GR-mediated activation at the level of chromatin remodelling and (4) how the role of these HAT complexes is integrated with other chromatin remodelling activities during GR-mediated gene activation. Our results suggest a model in which GR recruits the SAGA complex and that this contributes to chromatin remodelling via a mechanism involving the acetylation of histones. Furthermore, recruitment of the SWI/SNF remodelling complex also has a role in GR-mediated activation that is independent of the role of SAGA. These complexes are similar to analogous mammalian complexes and therefore these results are likely to be relevant to the human system.

Architectural principles for the structure and function of the glucocorticoid receptor tau 1 core activation domain.

A 58-amino acid region mediates the core transactivation activity of the glucocorticoid receptor tau1 activation domain. This tau1 core domain is unstructured in aqueous buffers, but in the presence of trifluoroethanol three alpha-helical segments are induced. Two of these putative structural modules have been tested in different combinations with regard to transactivation potential in vivo and binding capacity to the coactivators in vitro. The results show that whereas single modules are not transcriptionally active, any combination of two or three modules is sufficient, with trimodular constructs having the highest activity. However, proteins containing one, two, or three segments bind Ada2 and cAMP-response element-binding protein with similar affinity. A single segment is thus able to bind a target factor but cannot transactivate target genes significantly. The results are consistent with models in which activation domains are comprised of short activation modules that allow multiple interactions with coactivators. Our results also suggest that an increased number of modules may not result in correspondingly higher affinity but instead that the concentration of binding sites is increased, which gives rise to a higher association rate. This is consistent with a model where the association rate for activator-target factor interactions rather than the equilibrium constant is the most relevant measure of activator potency.

Comparison of nucleosome remodeling by the yeast transcription factor Pho4 and the glucocorticoid receptor.

Chromatin reorganization of the PHO5 and murine mammary tumor virus (MMTV) promoters is triggered by binding of either Pho4 or the glucocorticoid receptor (GR), respectively. In order to compare the ability of Pho4 and GR to remodel chromatin and activate transcription, hybrid promoter constructs were created by insertion of the MMTV B nucleosome sequence into the PHO5 promoter and then transformed into a yeast strain expressing GR. Activation of either Pho4 (by phosphate depletion) or GR (by hormone addition) resulted in only slight induction of hybrid promoter activity. However, simultaneous activation of both Pho4 and GR resulted in synergistic activation to levels exceeding that of the wild type PHO5 promoter. Under these conditions, Pho4 completely disrupted the nucleosome containing its binding site. In contrast, GR had little effect on the stability of the MMTV B nucleosome. A minimal transactivation domain of the GR fused to the Pho4 DNA-binding domain is capable of efficiently disrupting the nucleosome with a Pho4-binding site, whereas the complementary hybrid protein (Pho4 activation domain, GR DNA-binding domain) does not labilize the B nucleosome. Therefore, we conclude that significant activation by Pho4 requires nucleosome disruption, whereas equivalent transcriptional activation by GR is not accompanied by overt perturbation of nucleosome structure. Our results show that the DNA-binding domains of the two factors play critical roles in determining how chromatin structure is modified during promoter activation.

Recruitment of the SWI-SNF chromatin remodeling complex as a mechanism of gene activation by the glucocorticoid receptor tau1 activation domain.

The SWI-SNF complex has been shown to alter nucleosome conformation in an ATP-dependent manner, leading to increased accessibility of nucleosomal DNA to transcription factors. In this study, we show that the SWI-SNF complex can potentiate the activity of the glucocorticoid receptor (GR) through the N-terminal transactivation domain, tau1, in both yeast and mammalian cells. GR-tau1 can directly interact with purified SWI-SNF complex, and mutations in tau1 that affect the transactivation activity in vivo also directly affect tau1 interaction with SWI-SNF. Furthermore, the SWI-SNF complex can stimulate tau1-driven transcription from chromatin templates in vitro. Taken together, these results support a model in which the GR can directly recruit the SWI-SNF complex to target promoters during glucocorticoid-dependent gene activation. We also provide evidence that the SWI-SNF and SAGA complexes represent independent pathways of tau1-mediated activation but play overlapping roles that are able to compensate for one another under some conditions.

Activation domain-mediated targeting of the SWI/SNF complex to promoters stimulates transcription from nucleosome arrays.

The yeast SWI/SNF complex is required for the transcription of several yeast genes and has been shown to alter nucleosome structure in an ATP-dependent reaction. In this study, we show that the complex stimulated in vitro transcription from nucleosome templates in an activation domain-dependent manner. Transcription stimulation by SWI/SNF required an activation domain with which it directly interacts. The acidic activation domains of VP16, Gcn4, Swi5, and Hap4 interacted directly with the purified SWI/SNF complex and with the SWI/SNF complex in whole-cell extracts. The similarity of activation domain interactions and transcriptional stimulation between SWI/SNF and the SAGA histone acetyltransferase complex may account for their apparent overlapping functions in vivo.

Histone acetyltransferase complexes can mediate transcriptional activation by the major glucocorticoid receptor activation domain.

Previous studies have shown that the Ada adapter proteins are important for glucocorticoid receptor (GR)-mediated gene activation in yeast. The N-terminal transactivation domain of GR, tau1, is dependent upon Ada2, Ada3, and Gcn5 for transactivation in vitro and in vivo. Using in vitro techniques, we demonstrate that the GR-tau1 interacts directly with the native Ada containing histone acetyltransferase (HAT) complex SAGA but not the related Ada complex. Mutations in tau1 that reduce tau1 transactivation activity in vivo lead to a reduced binding of tau1 to the SAGA complex and conversely, mutations increasing the transactivation activity of tau1 lead to an increased binding of tau1 to SAGA. In addition, the Ada-independent NuA4 HAT complex also interacts with tau1. GAL4-tau1-driven transcription from chromatin templates is stimulated by SAGA and NuA4 in an acetyl coenzyme A-dependent manner. Low-activity tau1 mutants reduce SAGA- and NuA4-stimulated transcription while high-activity tau1 mutants increase transcriptional activation, specifically from chromatin templates. Our results demonstrate that the targeting of native HAT complexes by the GR-tau1 activation domain mediates transcriptional stimulation from chromatin templates.

Valine 571 functions as a regional organizer in programming the glucocorticoid receptor for differential binding of glucocorticoids and mineralocorticoids.

The glucocorticoid receptor (GR) interacts specifically with glucocorticoids, whereas its closest relative, the mineralocorticoid receptor (MR), interacts with both glucocorticoids and mineralocorticoids, such as aldosterone. To investigate the mechanism underlying the glucocorticoid/mineralocorticoid specificity of the GR, we used a yeast model system to screen for GR ligand-binding domain mutants, substituted with MR residues in the segment 565-574, that can be efficiently activated by aldosterone. In all such increased activity mutants, valine 571 was replaced by methionine, even though most mutants also contained substitutions of other residues with their MR counterparts. Further analysis in yeast and COS-7 cells has revealed that the identity of residue 571 determines the behavior of other MR substituted residues in the 565-574 segment. Generally, MR substitutions in this region are only consistent with aldosterone binding if residue 571 is also replaced with methionine (MR conformation). If residue 571 is valine (GR conformation), most other MR substitution mutants drastically reduce interaction with both mineralocorticoid and glucocorticoid hormones. Based on these functional data, we hypothesize that residue 571 functions as a regional organizer involved in discriminating between glucocorticoid and mineralocorticoid hormones. We have used a molecular model of the GR ligand-binding domain in an attempt to interpret our functional data in structural terms.

Identification and functional characterization of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae.

The so-called thioredoxin system, thioredoxin (Trx), thioredoxin reductase (Trr), and NADPH, acts as a disulfide reductase system and can protect cells against oxidative stress. In Saccharomyces cerevisiae, two thioredoxins (Trx1 and Trx2) and one thioredoxin reductase (Trr1) have been characterized, all of them located in the cytoplasm. We have identified and characterized a novel thioredoxin system in S. cerevisiae. The TRX3 gene codes for a 14-kDa protein containing the characteristic thioredoxin active site (WCGPC). The TRR2 gene codes for a protein of 37 kDa with the active-site motif (CAVC) present in prokaryotic thioredoxin reductases and binding sites for NADPH and FAD. We cloned and expressed both proteins in Escherichia coli, and the recombinant Trx3 and Trr2 proteins were active in the insulin reduction assay. Trx3 and Trr2 proteins have N-terminal domain extensions with characteristics of signals for import into mitochondria. By immunoblotting analysis of Saccharomyces subcellular fractions, we provide evidence that these proteins are located in mitochondria. We have also constructed S. cerevisiae strains null in Trx3 and Trr2 proteins and tested them for sensitivity to hydrogen peroxide. The Deltatrr2 mutant was more sensitive to H2O2, whereas the Deltatrx3 mutant was as sensitive as the wild type. These results suggest an important role of the mitochondrial thioredoxin reductase in protection against oxidative stress in S. cerevisiae.

Competition between thyroid hormone receptor-associated protein (TRAP) 220 and transcriptional intermediary factor (TIF) 2 for binding to nuclear receptors. Implications for the recruitment of TRAP and p160 coactivator complexes.

Transcriptional activation by nuclear receptors (NRs) involves the concerted action of coactivators, chromatin components, and the basal transcription machinery. Crucial NR coactivators, which target primarily the conserved ligand-regulated activation (AF-2) domain, include p160 family members, such as TIF2, as well as p160-associated coactivators, such as CBP/p300. Because these coactivators possess intrinsic histone acetyltransferase activity, they are believed to function mainly by regulating chromatin-dependent transcriptional activation. Recent evidence suggests the existence of an additional NR coactivator complex, referred to as the thyroid hormone receptor-associated protein (TRAP) complex, which may function more directly as a bridging complex to the basal transcription machinery. TRAP220, the 220-kDa NR-binding subunit of the complex, has been identified in independent studies using both biochemical and genetic approaches. In light of the functional differences identified between p160 and TRAP coactivator complexes in NR activation, we have attempted to compare interaction and functional characteristics of TIF 2 and TRAP220. Our findings imply that competition between the NR-binding subunits of distinct coactivator complexes may act as a putative regulatory step in establishing either a sequential activation cascade or the formation of independent coactivator complexes.

myc boxes, which are conserved in myc family proteins, are signals for protein degradation via the proteasome.

Cellular levels of the rapidly degraded c-myc protein play an important role in determining the proliferation status of cells. Increased levels of c-myc are frequently associated with rapidly proliferating tumor cells. We show here that myc boxes I and II, found in the N termini of all members of the myc protein family, function to direct the degradation of the c-myc protein. Both myc boxes I and II contain sufficient information to independently direct the degradation of otherwise stably expressed proteins to which they are fused. At least part of the myc box-directed degradation occurs via the proteasome. The mechanism of myc box-directed degradation appears to be conserved between yeast and mammalian cells. Our results suggest that the myc boxes may play an important role in regulating the level and activity of the c-myc protein.

Role of important hydrophobic amino acids in the interaction between the glucocorticoid receptor tau 1-core activation domain and target factors.

In this work, we determined how altered-function mutants affecting hydrophobic residues within the tau 1-core activation domain of the human glucocorticoid receptor (GR) influence its physical interaction with different target proteins of the transcriptional machinery. Screening of putative target proteins showed that the tau 1-core can interact with the C-terminal part of the CREB-binding protein (CBP). In addition, the previously identified interactions of the tau 1-core with the TATA-binding protein (TBP) and the Ada2 adaptor protein were localized to the C- and N-terminal regions of these proteins, respectively. A panel of mutations within the tau 1-core that either decrease or increase activation potential was used to probe the interaction of the tau 1-core domain with TBP, Ada2, and CBP. We found that the pattern of effects caused by the mutations was similar for each of the interactions and that the effects on binding generally reflected effects on gene activation potential. Thus, the predominant effect of the mutations appears to influence a property of the tau 1-core that is common to all three interactions, rather than properties that are differentially required by each of the target factor interactions, individually. Such a property could be the ability of the domain to adopt a folded conformation that is generally necessary for interaction with target factors. We have also shown that TBP, Ada2, and CBP can interact with both the tau 1-core and the GR ligand-binding domain, offering a possible mechanism for synergistic interaction between the tau 1-core and other receptor activation domains. However, other target proteins (e.g., RIP140, and SRC-1), which interact with the GR C terminus, did not show significant interactions with the tau 1-core under our conditions.

Involvement of the transcription factor IID protein complex in gene activation by the N-terminal transactivation domain of the glucocorticoid receptor in vitro.

HeLa cell nuclear extracts were used to study the mechanism of activation of RNA polymerase II-mediated transcription by the N-terminal transactivation domain (tau1) of the glucocorticoid receptor in vitro. When fused to the Gal4 DNA-binding domain, the tau1 domain activated transcription approximately 9-fold in HeLa nuclear extracts. Using heat treatment to inactivate transcription factor IID (TFIID) in the extract, it was shown that the addition of purified TFIID complex, but not the TATA-binding protein alone, was sufficient to restore this level of activation. The tau1 domain was shown to interact directly with the TFIID complex. This interaction was markedly reduced by a mutation in the tau1 domain that reduces its activity. Furthermore, the interaction was specific for the TFIID complex, since no interaction was seen with TFIIIB, an analogous protein complex involved in RNA polymerase III transcription. The tau1 domain was further shown to interact with the TATA-binding protein subunit of the TFIID complex. These results suggest a mechanism by which the GR tau1 domain might contribute to gene activation by recruitment of the TFIID complex to target promoters.

Rapid purification of glial cells using immunomagnetic separation.

By purifying glial cells from brain tissue containing a heterogeneous cell population, a number of interactions that define glial cell diversification and function within the central nervous system have been determined. The current methods for purifying glial cells, however, can be time consuming and costly. In the following study we have adapted the technique of immunomagnetic separation to separately enrich 0-2A progenitor cells and astrocytes from the rat central nervous system (CNS). In this procedure, tissue from the CNS was enzymatically dissociated and incubated in a primary antibody specific to a surface antigen found on the target cell type (e.g. A2B5 or RAN-2). The target cells were then immunologically coupled to magnetic beads, which were precoated with a secondary antibody specific to the primary, and then separated out from the heterogeneous cell population using a magnetic field. We found that the immunomagnetic separation procedure, which was completed within 2 h, produced a near pure population of glial cells (> 99%). This was confirmed by the absence of unbound cells in the bead-bound fraction. The identification and viability of bead-bound cells were established by culturing these cells and subsequently examining their morphology and antigenic expression. This study shows that glial cell types can be separated out of brain tissue to near purity using immunomagnetic separation. This simple procedure is reliable, inexpensive, and achieves levels of purity and viability comparable with currently available techniques of immunopanning and fluorescence-activated cell sorting, within a fraction of the time.

Role of the Ada adaptor complex in gene activation by the glucocorticoid receptor.

We have shown that the Ada adaptor complex is important for the gene activation capacity of the glucocorticoid receptor in yeast. The recently isolated human Ada2 protein also increases the potency of the receptor protein in mammalian cells. The Ada pathway is of key significance for the tau1 core transactivation domain (tau1c) of the receptor, which requires Ada for activity in vivo and in vitro. Ada2 can be precipitated from nuclear extracts by a glutathione S-transferase-tau1 fusion protein coupled to agarose beads, and a direct interaction between Ada2 and tau1c can be shown by using purified proteins. This interaction is strongly reduced by a mutation in tau1c that reduces transactivation activity. Mutations affecting the Ada complex do not reverse transcriptional squelching by the tau1 domain, as they do for the VP16 transactivation domain, and thus these powerful acidic activators differ in at least some important aspects of gene activation. Mutations that reduce the activity of the tau1c domain in wild-type yeast strains cause similar reductions in ada mutants that contain little or no Ada activity. Thus, gene activation mechanisms, in addition to the Ada pathway, are involved in the activity of the tau1c domain.