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The activation of the transcription factor hypoxia-inducible factor (HIF)-1 is associated with an aggressive tumor phenotype. HIF-1 is regulated by hydroxylase enzymes that require vitamin C (ascorbate) as cofactor and analyses of clinical tumor samples and dietary supplementation studies in mice have demonstrated an inverse correlation between tumor ascorbate levels and HIF-1 activity. In this study we investigated whether pharmacological doses of ascorbate can affect tumor ascorbate levels and exert an anti-tumor effect in tumor-bearing Gulo-/- mice, a model of human ascorbate dependency. Ascorbate concentrations peaked within an hour after a single intra-peritoneal injection (1g/kg), with millimolar levels measureable in plasma, and increased levels in liver and tumor tissues. Plasma and liver ascorbate returned to baseline within 16h, but tumor levels remained elevated for 48h. Modelling ascorbate uptake and stability in tumors suggests that increased stability under hypoxia may account for its prolonged presence within the tumor tissue. The expression of HIF-1 and its target proteins was down-regulated during the first 4h post-injection, and increased again after 16–48h. Increased tumor ascorbate levels could be maintained with repeated daily or alternate day administration. HIF-1 and vascular endothelial growth factor proteins were inversely correlated with tumor ascorbate and were significantly down-regulated in tumors when ascorbate was administered daily. Increased tumor ascorbate was associated with reduced microvessel density, tumor hypoxia and tumor growth. These results support the suppression of the hypoxic response as being a possible anti-tumor activity of ascorbate that may be useful in a clinical setting.

Adverse psychological and physical impacts have been related to experience of detriment arising from sex discrimination. In the present study detriment was operationalized in terms of differential career progress, unequal access to organizational benefits and experience of sexual harassment. A national UK sample of 1802 police women, 510 police men and 164 civilian women took part in the study. Results revealed gender differences in exposure to detriment and the suffering of associated adverse impacts. Subjective rating of impacts by police women revealed the greatest impact to be associated with frustrated career progression and the least from experience of sexual harassment. A multiple regression analysis suggested the reverse order when predicting elevated GHQ(28) scores for women officers. This is accounted for by cultural factors, which are also proposed to be influential in rates of reporting sources of discrimination.

Mycobacterium tuberculosis infection continues to cause substantial human suffering. New chemotherapeutic strategies, which require insight into the pathways essential for M. tuberculosis pathogenesis, are imperative. We previously reported that depletion of the CarD protein in mycobacteria compromises viability, resistance to oxidative stress and fluoroquinolones, and pathogenesis. CarD associates with the RNA polymerase (RNAP), but it has been unknown which of the diverse functions of CarD are mediated through the RNAP; this question must be answered to understand the CarD mechanism of action. Herein, we describe the interaction between the M. tuberculosis CarD and the RNAP β subunit and identify point mutations that weaken this interaction. The characterization of mycobacterial strains with attenuated CarD/RNAP β interactions demonstrates that the CarD/RNAP β association is required for viability and resistance to oxidative stress but not for fluoroquinolone resistance. Weakening the CarD/RNAP β interaction also increases the sensitivity of mycobacteria to rifampin and streptomycin. Surprisingly, depletion of the CarD protein did not affect sensitivity to rifampin. These findings define the CarD/RNAP interaction as a new target for chemotherapeutic intervention that could also improve the efficacy of rifampin treatment of tuberculosis. In addition, our data demonstrate that weakening the CarD/RNAP β interaction does not completely phenocopy the depletion of CarD and support the existence of functions for CarD independent of direct RNAP binding.

Blind-ended, hollow fibers mounted on a pulmonary artery catheter may allow O 2 and CO 2 transfer in the vena cava, right ventricle, and pulmonary artery. The effects of fiber length, manifold number, and gas oscillation on mass and momentum transfer with water perfusate using mass spectrometry and mass flow controllers were studied. Manifolds with 112-196 microporous polypropylene fibers were mounted on 8 Fr multiple lumen, commercially available pulmonary artery catheters. Fiber lengths varied from 0.5 to 16 cm and surface areas from 7 to 220 cm 2 . Prototypes with 2 cm long fibers were constructed with 1-15 manifolds. A two manifold prototype with 8 cm long fibers and a surface area of 378 cm 2 was also studied. The transfer failed to scale with manifold number because the steady gas flow was maldistributed to the manifolds. Oscillating gas pressures from 780 to 76 mmHg absolute at a rate of 40 cycles/min increased CO 2 transfer up to 15-fold and O 2 transfer up to 2.5-fold. Oscillation also corrected the maldistribution. Optimal fiber lengths of 3 and 1 cm for O 2 and CO 2 , respectively, were seen with steady gas flow, and 8 cm for both with oscillatory gas flow

This article describes a decade-long partnership between the Prevention Research Center at Penn State and the Pennsylvania Commission on Crime and Delinquency. This partnership has evolved into a multi-agency initiative supporting the implementation of nearly 200 replications of evidence-based prevention and intervention programs, and a series of studies indicating a significant and sustained impact on youth outcomes and more efficient utilization of system resources. We describe how the collaboration has developed into a sophisticated prevention support infrastructure, discuss the partnership and policy lessons learned throughout this journey, and identify remaining issues in promoting this type of research-policy partnership.

Teachers are always searching for fresh ideas that address children's growth needs. Ideas that provide learning opportunities which are developmentally appropriate are particularly welcomed. using play as a medium for facilitating literacy growth is one such opportunity. We know from current theory, research and practice that sociodramatic play contributes to children's learning. Its importance in the development of literacy skills has also been investigated and affirmed.

Structural variants of the hydrophobic side chain («C region») of the capsaicin molecule have been incorporated into a series of vanillylamides and vanillylthioureas. These compounds have been tested in an in vitro assay for agonism ( 45 Ca 2+ influx into dorsal root ganglia neurones), previously shonvn to be predictive of analgesic activity. The results of this study have established the requirement for a hydrophobic substituent of limited size (molar refractivity, MR

A series of analogues of capsaicin, the pungent principle of chilli peppers, was synthesized and tested in assays for capsaicin-like agonism in vitro. The results of these assays were compared with activities in an acute nociceptive model and a correlation was observed which established that the results of these in vitro assays were predictive of analgesia. Using a modular approach the structure-activity profile of specific regions of capsaicin congeners was established using an in vitro assay measuring 45Ca2+ uptake into neonatal rat dorsal root ganglia neurones. Substituted benzylnonanamides 2a-z and N-octyl-substituted phenylacetamides 4a-v were made to test the requirements for activity in the aromatic "A-region" of the molecule. Compounds with the natural substitution pattern (2b and 4c) and the corresponding catechols (2i and 4g) were the most potent, although the catechols were less potent in vivo. Other substitution patterns have reduced activity. These results have established stringent structural requirements for capsaicin-like activity in this part of the molecule.

Forensic psychology has developed and extended from an original, narrow focus on presenting evidence to the courts to a wider application across the whole span of civil and criminal justice, which includes dealing with suspects, offenders, victims, witnesses, defendants, litigants and justice professionals. This Handbook provides an encyclopedic-style source regarding the major concerns in forensic psychology. It is an invaluable reference text for practitioners within community, special hospital, secure unit, prison, probation and law enforcement forensic settings, as well as being appropriate for trainees and students in these areas. It will also serve as a companion text for lawyers and psychiatric and law enforcement professionals who wish to be apprised of forensic psychology coverage. Each entry provides a succinct outline of the topic, describes current thinking, identifies relevant consensual or contested aspects and alternative positions. Readers are presented with key issues and directed towards specialized sources for further reference.

The bacterial RNA polymeras holoenzyme consists of a catalytic core enzyme in complex with a sigma factor that is required for promoter-specific transcription initiation. During initiation, members of the sigma(70) family of sigma factors contact two conserved promoter elements, the -10 and -35 elements, which are separated by approximately 17 base pairs (bp). sigma(70) family members contain four flexibly linked domains. Two of these domains, sigma(2) and sigma(4), contain determinants for interactions with the promoter -10 and -35 elements respectively. sigma(2) and sigma(4) also contain core-binding determinants. When bound to core the inter-domain distance between sigma(2) and sigma(4) matches the distance between promoter elements separated by approximately 17 bp. Prior work indicates that during early elongation the nascent RNA-assisted displacement of sigma(4) from core can enable the holoenzyme to adopt a configuration in which sigma(2) and sigma(4) are bound to 'promoter-like' DNA elements separated by a single base pair. Here we demonstrate that holoenzyme can also adopt configurations in which sigma(2) and sigma(4) are bound to 'promoter-like' DNA elements separated by 0, 2 or 3 bp. Thus, our findings suggest that displacement of sigma(4) from core enables the RNA polymerase holoenzyme to adopt a broad range of 'elongation-specific' configurations.

A quantitative flow cytometry method for the analysis of membrane-associated human chorionic gonadotropin (hCG), its subunits, and fragments on human cancer cells was developed using a double-antibody reaction; a flow cytometry with a 2-W argon laser, standard settings, and filters for fluorescein isothiocyanate use; commercially available software; and the ectopic hCG producer CCL 2 HeLa cells from the American Type Culture Collection (ATCC) as a cell control to standardize the reagents and for overall quality control. Twenty-two monoclonal antibodies (MoAb) and immunoglobulin G fractions from three rabbit polyclonal antisera were tested for effects of antibody concentration (titration), reproducibility at different levels of epitope expression, and variability of epitope expression to select appropriate primary antibodies. Based on the results of the various tests, three polyclonal immunoglobulin G antibodies and a panel of nine MoAb directed to epitopes located in five different regions on the hCG molecule were selected as first antibodies. Their specificity was determined by using two unrelated MoAb of the same isotype at the same concentration to replace the primary MoAb and by a competition experiment. The unrelated MoAb also were used for the selection of the appropriate control fluorescence profile needed for the software. The unique characteristics of this method were: the use of living cells, standardized reagents, internal and external quality control, and the highest sensitivity, which could detect as few as 10(3) molecules of fluorochrome per cell. Serial analyses of the ATCC CCL 2 HeLa cells and two of its variants and of the eutopic hCG producer JEG-3 choriocarcinoma cells revealed the expression of membrane-associated epitopes of intact hCG, its subunits, and fragments by a high percentage of the cells, indicating that the expression of these sialoglycoproteins by these two different types of cancer cells is a common phenotypic characteristic.

The expression of human chorionic gonadotropin (hCG), its subunits, and fragments on the cell membrane of cultured human cancer cells was investigated using a flow cytometric method. This method uses living cells; a double-antibody reaction; a flow cytometer with an argon laser, standard settings, and filters for fluorescein isothiocyanate; commercially available software; the American Type Culture Collection (ATCC) CCL 2 HeLa cell line as cell control and overall quality control; polyclonal rabbit antisera raised against the hCG dimer, its alpha subunit (hCG alpha), and its beta subunit (hCG beta); and a panel of monoclonal antibodies (MoAb) recognizing different epitopes on the intact hCG molecule, its subunits, and fragments. The purified immunoglobulin G fractions from the polyclonal antisera were used to estimate the total expression of the membrane-associated glycoproteins; the MoAb were used to detect the expression of epitopes of the hCG dimer, its subunits, and fragments. The results of the analyses done on cells from 74 established cancer cell lines of different types and origins (including 52 carcinomas, 10 sarcomas, 4 leukemias, 6 lymphomas, and 2 retinoblastomas) showed variable degrees of reactivity in a great percentage of cells in all cell lines studied with MoAb directed against different conformational epitopes of intact hCG (hCG-holo), hCG beta, hCG beta-free, the carboxy terminal peptide (CTP) of hCG beta, and an epitope of hCG alpha. The expression of the membrane-associated epitopes of hCG and its subunits was found to be a phenotypic marker characteristic of all evaluated cultured human cancer cell lines, irrespective of their type or origin. There were, however, quantitative and qualitative differences in the expression of the different epitopes. Thus, hCG beta, free and as part of hCG-holo, recognized by the MoAb against hCG beta-CTP, was expressed by a high percentage of cells of most cell lines. There was great variability in the expression of hCG-holo, recognized by MoAb B109. For this reason some groups of cancers expressed larger amounts of incompetent hCG alpha and/or hCG beta than others. Cell lines derived from adenocarcinomas of the lung were the only exception to this general finding; the expression of small amounts of hCG-holo was caused by a low degree of hCG alpha synthesis.

International audience; The Bacillus subtilis Spx protein is a global transcription factor that interacts with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) and regulates transcription of genes involved in thiol-oxidative stress, sporulation, competence, and organosulfur metabolism. Here we determined the X-ray crystal structure of the Spx/alphaCTD complex from an entirely new crystal form than previously reported [Newberry, K.J., Nakano, S., Zuber, P., Brennan, R.G., 2005. Crystal structure of the Bacillus subtilis anti-alpha, global transcriptional regulator, Spx, in complex with the alpha C-terminal domain of RNA polymerase. Proc. Natl. Acad. Sci. USA 102, 15839-15844]. Comparison of the previously reported sulfate-bound complex and our sulfate-free complex reveals subtle conformational changes that may be important for the role of Spx in regulating organosulfur metabolism.

(1991). Teaching and promoting smoking cessation counseling in primary care residencies: Description of a method. Teaching and Learning in Medicine: Vol. 3, No. 1, pp. 20-27.

Study objective:To evaluate the effectiveness of two teaching interventions to increase residents’ performance of smoking cessation counseling. Design:Randomized controlled factorial trial. Setting:Eleven residency programs, in internal medicine (six), family medicine (three), and pediatrics (two). Programs were located in three university medical centers and four university-affiliated community hospitals. Participants:261 residents who saw ambulatory care patients at least one half-day per week, and 937 returning patients aged 17 to 75 years who reported having smoked five or more cigarettes in the preceding seven days. Of the 937, 843 were eligible for follow-up, and 659 (78%) were interviewed by phone at six months. Interventions:Two interventions (tutorial and prompt) and four groups. The tutorial was a two-hour educational program in minimal-contact smoking cessation counseling for residents. The prompt was a chart-based reminder to assist physician counseling. One group of residents received the tutorial; one, the prompt; and one, both. A fourth group received no intervention. Measurement and results:Six months after the intervention, physician self-reports showed that residents in the tutorial + prompt and tutorial-only groups had used more counseling techniques (1.5–1.9) than had prompt-only or control residents (0.9). Residents in all three intervention groups advised more patients to quit smoking (76–79%) than did control group residents (69%). The tutorial had more effect on counseling practices than did the prompt. Physician confidence, perceived preparedness, and perceived success followed similar patterns. Exit interviews with 937 patients corroborated physician self-reports of counseling practices. Six months later, self-reported and biochemically verified patient quitting rates for residents in the three intervention groups (self-reported: 5.3–8.2%; biochemically verified: 3.4–5.7%) were higher than those for residents in the control group (self-reported: 5.2%; biochemically verified: 1.7%), though the differences were not statistically significant. Conclusion:A simple and feasible educational intervention can increase residents’ smoking cessation counseling.

Background: Maternal mental health, in particular depression, has been found to negatively impact mother-child interaction, attachment, stimulation, growth, and many important aspects of development in the young child. These early deficits if sustained and unattended may have negative immediate and long-term consequences on the outcomes in the child. The study aimed to assess psychological distress and postpartum depression in mothers, and their relationship to the mother-child interaction. Methodology: This is a descriptive clinic-based study. Eligible and consenting mothers are attending the child immunization clinic in the Lagos University Teaching Hospital, Lagos, Nigeria participated. Consecutive mothers completed the interview questionnaires independently while those who were not literate had the questionnaires administered by trained interviewers. Instruments used were a sociodemographic proforma, the General Health Questionnaire-12, Mother and Infant Attachment Scale (MIAS), and the Zung Depression Scale. Results: In total, ninety-eight women were enrolled, 66.3% were aged between 26 and 35 years, and mean age of 30.9 years (±5.1 standard deviation). Most were aged between 26 and 35 years (66.3%). Over 90% had at secondary school education or more. Over a 10 th (13.3%) was unemployed and 96% married. The children were aged between 6 weeks and 1 year, males (63.1%), and females (46.9%), and the majority were born by spontaneous vaginal delivery (82.7%). A 10 th (10.2%) of the women had probable psychiatric morbidity, 14.3% had scores suggestive of postpartum depression, and 18 (16.3%) scored below average attachment in interaction with their children on the MIAS. There was an association found between reduced maternal-child attachment interaction and maternal depression (P ≤ 0.05). Conclusions: Emotional disorders, such as depression, in mothers can be associated reduced maternal-child interaction. It is important that integrated mental health care services be offered to women.

This paper describes an empirical study (n = 954) of occupational stress conducted in a large provincial English police force. Conceptual distinctions were made between stressors, i.e., potential external sources of adverse reactions; felt stress, i.e., self-perceived negative impact; and distress, i.e., self-reported symptoms of anxiety, depression, social dysfunction, and somatic disturbance. Findings indicates that overall exposure rates are highest for organizational and management stressors rather than routine operational duties. Differential rates of exposure, felt stress, and distress are reported for different ranks of officers. The paper confirms the nature of postulated sources of police stress from earlier studies and extends analysis to differentiate between exposure to stressors and experience of distress.

If public health research does not progress beyond descriptive research to testing the effectiveness of interventions that can provide causal data, its contribution to evidence-based practice will not be realized. This paper examined the number and percentage of public health research publications over three time periods (1987-1988, 1997-1998, 2005-2006) for three preventive health issues: tobacco use, alcohol use, and inadequate physical activity.A computer-based literature search was conducted, using the Medline and PsycINFO databases. A random sample of 1000 abstracts for each preventive health issue was examined per time period. The abstracts were first categorized as public health or not, and then as data-based or non-data-based public health research. Data-based publications were classified according to research type as measurement-, descriptive/epidemiologic-, or intervention-oriented. Data analysis occurred in 2007.The number of data-based public health research publications examined for each topic ranged between 180 and 346 in 1987-1988, 199 and 322 in 1997-1998, and 302 and 364 in 2005-2006. Despite a significant increase over time in the percentage of intervention publications for tobacco (from 10% in 1987-1988 to 18% in 2005-2006, p=0.008), the majority of publications in all three time periods were descriptive/epidemiologic for all topics (62%-87% in 1987-1988, 64%-85% in 1997-1998, 78%-79% in 2005-2006). There were fewer measurement (3%-7% in 1987-1988, 2%-6% in 1997-1998, 4%-10% in 2005-2006) or intervention publications (9%-31% in 1987-1988, 10%-30% in 1997-1998, 12%-18% in 2005-2006).Descriptive research does not provide optimal evidence for how to reduce preventable illness. Reasons for the lack of measurement and intervention public health research are explored.

The Escherichia coli Rsd protein binds tightly and specifically to the RNA polymerase (RNAP) sigma(70) factor. Rsd plays a role in alternative sigma factor-dependent transcription by biasing the competition between sigma(70) and alternative sigma factors for the available core RNAP. Here, we determined the 2.6 A-resolution X-ray crystal structure of Rsd bound to sigma(70) domain 4 (sigma(70)(4)), the primary determinant for Rsd binding within sigma(70). The structure reveals that Rsd binding interferes with the two primary functions of sigma(70)(4), core RNAP binding and promoter -35 element binding. Interestingly, the most highly conserved Rsd residues form a network of interactions through the middle of the Rsd structure that connect the sigma(70)(4)-binding surface with three cavities exposed on distant surfaces of Rsd, suggesting functional coupling between sigma(70)(4) binding and other binding surfaces of Rsd, either for other proteins or for as yet unknown small molecule effectors. These results provide a structural basis for understanding the role of Rsd, as well as its ortholog, AlgQ, a positive regulator of Pseudomonas aeruginosa virulence, in transcription regulation.

Our laboratory has refined the technique for isolating primary cultures of normal human ovarian surface epithelial (OSE) cells by combining two different protocols involving the enzymatic and mechanical removal of OSE cells from ovarian biopsies. A simple protocol of obtaining primary epithelial ovarian cancer (EOC) cells from the ascites fluid removed from patients with high-grade ovarian cancer is also described. These methods allow for the direct application of many molecular and cellular analyses of normal versus cancer cells isolated freshly from patients, with the added potential for retrospective analyses of archived cells and tissues. Thus, we have included optional steps for the immediate preparation of ascites-derived EOC cells to be used for subsequent cytological analyses. Initial isolation of OSE or EOC cells can be completed in 1 h, and primary cells are further expanded in culture for several weeks.

Nat. Protoc. 1, 2643–2649 (2007); doi:10.1038/nprot.2006.328; published online 11 January 2007; corrected after print 5 August 2015 In the version of this article initially published, in the Reagent Setup section, it was stated that 50% v/v Percoll should be made by diluting Percoll in 10× PBS at a 1:1 v/v ratio.

Refugees in Nairobi are simultaneously accepted, needed, and depended on as key actors in the local economy and rejected as illegal foreigners, accused of compounding problems of joblessness and poverty in the city. Although the vast majority of Nairobi’s refugees live illegally in the city and face widespread institutionalized discrimination, they have managed nonetheless to forge multi-stranded social and economic relations that link together their countries of origin, countries of asylum, and countries of resettlement (Horst 2003; 2004). These transnational networks have helped many urban refugees become successful entrepreneurs and prominent businessmen, particularly in the informal marketplace. Contrary to the official state position and the xenophobic local belief that refugees are a drain on limited national resources, urban refugees, stemming from throughout the Horn, Central, and East Africa, are largely self-sufficient. Firmly entrenched in Eastleigh, the heart of the African immigrant community in Nairobi, these transnational migrants have cultivated an ability to operate successfully within a socially hostile and economically depressive environment.

A combined structural, functional, and genetic approach was used to investigate inhibition of bacterial RNA polymerase (RNAP) by sorangicin (Sor), a macrolide polyether antibiotic. Sor lacks chemical and structural similarity to the ansamycin rifampicin (Rif), an RNAP inhibitor widely used to treat tuberculosis. Nevertheless, structural analysis revealed Sor binds in the same RNAP beta subunit pocket as Rif, with almost complete overlap of RNAP binding determinants, and functional analysis revealed that both antibiotics inhibit transcription by directly blocking the path of the elongating transcript at a length of 2-3 nucleotides. Genetic analysis indicates that Rif binding is extremely sensitive to mutations expected to change the shape of the antibiotic binding pocket, while Sor is not. We suggest that conformational flexibility of Sor, in contrast to the rigid conformation of Rif, allows Sor to adapt to changes in the binding pocket. This has important implications for drug design against rapidly mutating targets.

Self-immolation among young Muslim women in parts of the Middle East and Central Asia increasingly is becoming a cause of death and disability. Very little is known about this phenomenon. Thus, in this article we (1) examine, from the perspective of Muslim culture, the phenomenon of self-immolation among young Islamic women in the Muslim countries of Central Asia and the Middle East; (2) describe current international activities regarding Islamic female self-immolation; and (3) discuss the implications of Islamic female self-immolation for global women's heath research and practice.

Proprotein convertases (PC) are a family of serine endoproteases that play important roles in regulating cell function by converting proproteins to biologically active molecules. Several lines of evidence suggest that overexpression of PCs contributes to tumor formation and progression in various types of cancer. In this study, we examined PC expression in six normal ovarian surface epithelium (OSE) cultures, nine primary ovarian cancer (OC) cultures, and three established OC cell lines (Hey, HeyC2, and OCC-1). Our results show that furin and PC7 expression in OC cells was comparable to that in normal OSE. However, PACE4 expression was greatly reduced in all OC samples studied. PACE4 promoter activity was measured in HeyC2 and OCC-1 cells using transiently transfected luciferase reporter plasmids. Both cell lines supported PACE4 promoter activity, showing that the transcription factors critical for PACE4 expression are present in OC cells. The observation that established OC cell lines have reduced PACE4 expression, but maintained the ability to support PACE4 promoter activity, led to the hypothesis that reduced expression may be due to epigenetic modification of the PACE4 gene, such as DNA methylation and histone deacetylation. Methylation analysis of 79 CpG dinucleotides within the PACE4 promoter and exon I (-196/+340) revealed that the percentage of methylated cytosine nucleotides was 8-9% in normal OSE, but 58-93% in OC cells. Treatment with the demethylating agent 5-aza-2'-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A greatly increased PACE4 expression in OC cells. These data suggest that the reduction of PACE4 expression in OC cells is caused, in part, by DNA hypermethylation and histone deacetylation.

Instilling and cultivating trust with clients, patients, and customers has become an increasingly important for highly expert service providers. We advance understanding of how service providers, s...

Bacterial σ factors combine with the catalytic core RNA polymerase to direct the process of transcription initiation through sequence-specific interactions with the −10 and −35 elements of promoter DNA. In the absence of core RNA polymerase, the DNA-binding function of σ is autoinhibited by its own N-terminal 90 amino acids (region 1.1), putatively by a direct interaction with conserved region 4.2, which binds the −35 promoter element. In the present work, this mechanism of autoinhibition was studied by using a combination of NMR spectroscopy and segmental isotopic labeling of a σ 70 -like subunit from Thermotoga maritima . Our data argue strongly against a high-affinity interaction between these two domains. Instead we suggest that autoinhibition of DNA binding occurs through an indirect steric and/or electrostatic mechanism. More generally, the present work illustrates the power of segmental isotopic labeling for probing molecular interactions in large proteins by NMR.

Cell type-specific transcription during Bacillus sporulation is established by sigmaF. SpoIIAB is an anti-sigma that binds and negatively regulates sigmaF, as well as a serine kinase that phosphorylates and inactivates the anti-anti-sigma SpoIIAA. The crystal structure of sigmaF bound to the SpoIIAB dimer in the low-affinity, ADP form has been determined at 2.9 A resolution. SpoIIAB adopts the GHKL superfamily fold of ATPases and histidine kinases. A domain of sigmaF contacts both SpoIIAB monomers, while 80% of the sigma factor is disordered. The interaction occludes an RNA polymerase binding surface of sigmaF, explaining the SpoIIAB anti-sigma activity. The structure also explains the specificity of SpoIIAB for its target sigma factors and, in combination with genetic and biochemical data, provides insight into the mechanism of SpoIIAA anti-anti-sigma activity.

The sigma subunit is the key regulator of bacterial transcription. Proteolysis of Thermus aquaticus sigma(A), which occurred in situ during crystallization, reveals three domains, sigma(2), sigma(3), and sigma(4), connected by flexible linkers. Crystal structures of each domain were determined, as well as of sigma(4) complexed with -35 element DNA. Exposed surfaces of each domain are important for RNA polymerase binding. Universally conserved residues important for -10 element recognition and melting lie on one face of sigma(2), while residues important for extended -10 recognition lie on sigma(3). Genetic studies correctly predicted that a helix-turn-helix motif in sigma(4) recognizes the -35 element but not the details of the protein-DNA interactions. Positive control mutants in sigma(4) cluster in two regions, positioned to interact with activators bound just upstream or downstream of the -35 element.

Disruptions in TGF beta signaling have been implicated in various human cancers, including ovarian cancer. Our goal was to determine whether ovarian cancer cells isolated from patient ascites fluid were growth inhibited by TGF beta 1 treatment and further characterize the expression and activity profile of TGF beta/Smad signaling components in human ovarian cancer cells. We found that 9 of 10 primary cultures of ovarian cancer cells (OC2-10) were growth inhibited by 16 pM TGF beta 1. One primary ovarian cancer sample (OC1) and the established ovarian cancer cell lines CaOV3 and SkOV3 continued to grow in the presence of TGF beta 1. All cells expressed components of the TGF beta/Smad signaling pathway including TGF beta 1, T beta RI, T beta RII, Smad2, -3, -4, and Smad anchor for receptor activation. Although OC1, CaOV3, and SkOV3 are not growth inhibited by TGF beta 1, they can transmit the TGF beta 1 signal to turn on a transfected TGF beta/Smad reporter gene, p3TP.lux. In addition, all cells up-regulate the endogenous TGF beta target genes Smad7 and PAI-1. p15(Ink4B) mRNA is also up-regulated with TGF beta 1 treatment in OC2-9, whereas the p15(Ink4B) gene has been deleted in OC1, CaOV3, and SkOV3 cells. Homozygous deletion of p15(Ink4B) may account for TGF beta resistance in some populations of ovarian cancer cells. Our data demonstrate that the TGF beta/Smad signaling pathway remains functional in human ovarian cancer cells and suggest that if abnormalities exist in the cellular response of TGF beta signals, they must lie downstream of the Smad proteins.

Humans are unable to synthesise ascorbate (vitamin C) due to a mutation in the gene for gulono-lactone oxidase (Gulo), the terminal enzyme in the synthesis pathway. Ascorbate is a vital micronutrient required for many biological functions, including its action as a vital cofactor for Cu- and Fe-containing enzymes. As enzyme cofactor for HIF-hydroxylases, it is important for the regulation of hypoxia-inducible factor-1 (HIF-1), which governs cancer growth and spread. A lack of ascorbate increases both the level and activity of HIF-1 in cell culture. This study aimed to restore ascorbate synthesis to human cells and to determine the effect of intracellular ascorbate biosynthesis on HIF-1 levels. HepG2 cells were gene modified using Lipofectamine LTX with a plasmid encoding the mouse Gulo cDNA and a control plasmid encoding the green fluorescent protein (GFP). Modified HepG2 cells were tested for genomic incorporation by PCR, for Gulo enzyme production by Western blotting, and ascorbate synthesis by high performance liquid chromatography with electrochemical detection (HPLC-ECD). Protein levels of HIF-1alpha in hypoxic cells were measured by Western blot analysis. Parental HepG2 cells accumulated a mean of 120 nmol ascorbate per 1 million cells when loaded for 24h with 0.5 mM ascorbate. Gene-modified HepG2Gulo cells demonstrated a change in morphology and an increase in adherence, compared to HepG2GFP and parental HepG2 cells. A PCR-positive stable clone was able to synthesise ascorbate but only when the Gulo substrate, L-gulono-1,4-lactone, was supplied. Intracellular ascorbate levels then reached 5% of saturation levels. Hypoxic HIF-1 accumulation was reduced by addition of gulonolactone to this clone. However, gulonolactone also reduced HIF-1 levels in parental HepG2 cells, suggesting that it may similarly act as cofactor for the HIF-hydroxlases. This study indicated that the ascorbate biosynthesis pathway in human cells is significantly modified and may contain numerous non-functional members besides gulono-lactone oxidase. Future biochemical studies will determine which other enzymes are non-functional. Citation Format: Teresa Flett, Elizabeth Campbell, Elisabeth Phillips, Margreet CM Vissers, Gabi U. Dachs. Does gene transfer of gulono-lactone oxidase into human hepatocellular carcinoma cells restore ascorbate biosynthesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 727. doi:10.1158/1538-7445.AM2014-727

Background: Hypoxia-inducible factor-1 (HIF-1) upregulates the expression of hundreds of genes involved in adaption of tumours to the hypoxic microenvironment. HIF-1 levels and transcriptional activity are regulated by hydroxylase enzymes, which require ascorbate as a cofactor. Adequate supplementation of cells with ascorbate has been shown to reduce HIF-1 activation in vitro, and we hypothesise that a similar activity could affect tumour growth in vivo. In this study we investigated the effect of dietary ascorbate on tumour growth in Gulo-/- mice, a model of the human ascorbate deficiency condition. Methods: C57/Bl6 Gulo-/- mice were supplemented with 3300 mg/L, 330 mg/L or 33mg/L of ascorbate in their drinking water for one month before subcutaneous, syngeneic tumour implantation with either B16-F10 melanoma, or Lewis lung carcinoma (LL/2), and then for duration of the experiment. Levels of HIF-1α, and its targets, carbonic anhydrase-IX (CA-IX), and glucose transporter-1 (GLUT-1) were analysed by western blotting, and vascular endothelial growth factor (VEGF) levels by ELISA. Ascorbate concentrations of tissue, plasma and tumours were measured using high pressure liquid chromatography with electrochemical detection (HPLC-EC). Results: Across all organs and plasma samples, ascorbate content was in the physiological range (up to levels seen in wild-type mice) and was correlated with dietary ascorbate intake. Tumour ascorbate also reflected ascorbate intake and optimal ascorbate levels (3300 mg/L) significantly increased the time for LL/2 tumours to reach 200 mm3 (lag growth) (13.3±2.6 days), compared to these same tumours in mice supplemented with either 330 mg/L (9.1±2.6 days) or 33 mg/L (9.8±1.39 days) of ascorbate (p Conclusions: The data from the current study support the hypothesis that restoration of optimal intracellular ascorbate levels is associated with reduced HIF-1 levels and slower tumour growth. This data may have implications for the management of cancer, although clinical trials are required to assess whether human tumour levels of ascorbate can similarly be manipulated. Citation Format: Elizabeth J. Campbell, Stephanie M. Bozonet, Bridget A. Robinson, Margreet CM Vissers, Gabi U. Dachs. Restoring physiological levels of ascorbate alleviates HIF-1 activation and reduces tumour growth in Gulo-/- mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 229. doi:10.1158/1538-7445.AM2014-229

Bacterial DNA-dependent RNA polymerase (RNAP) has subunit composition β′βα I α II ω. The role of ω has been unclear. We show that ω is homologous in sequence and structure to RPB6, an essential subunit shared in eukaryotic RNAP I, II, and III. In Escherichia coli , overproduction of ω suppresses the assembly defect caused by substitution of residue 1362 of the largest subunit of RNAP, β′. In yeast, overproduction of RPB6 suppresses the assembly defect caused by the equivalent substitution in the largest subunit of RNAP II, RPB1. High-resolution structural analysis of the ω–β′ interface in bacterial RNAP, and comparison with the RPB6–RPB1 interface in yeast RNAP II, confirms the structural relationship and suggests a “latching” mechanism for the role of ω and RPB6 in promoting RNAP assembly.

DNA-dependent RNA polymerases (RNAP) play a central role in transcription and therefore are a major regulatory target for gene expression. The regulation of prokaryotic transcription involves several protein-protein interactions between the different subunits of the RNAP complex as well as between several transcription factors. Understanding how these regulatory mechanisms work is crucial in order to open new strategies for targeting gene expression in pathogenic bacteria. Prokaryotic core RNAPs contain β, β′, and two a subunits, and are fully active in RNA polymerization. However, they are incapable of promoter recognition and specific initiation. Specific initiation of transcription depends on the binding of σ factors to the core RNAP to form the RNAP holoenzyme. The most abundant σ factor in E. coli is σ70. Sequence comparisons among different σ70-like factors has shown that they are comprised of four highly homologous regions [1]. Regions 2 and 4.2 have been shown to interact with the -10 and -35 box of the promoter region, respectively. Nonetheless, in the absence of the core RNAP, the σ70-like factors are unable to recognize the promoter sequence [2]. Region 1.1 has been shown to inhibit the ability by σ70-like factors to recognize the promoter DNA region [2]. Here, we have used segmental isotopic labeling techniques [3] to selectively label region 4.2 of σA (a homologous σ-factor from T. maritima) with 15N, 2H and 13C. This novel methodology has been used used in combination with NMR chemical shift perturbation studies [4] to study the interaction between these two regions.

Capsaicin and analogues are valuable analgesic agents when administered to mammals, including humans. However, their pungency and the effects on the cardiovascular and respiratory systems through their general activation of small calibre (nociceptive) primary afferents severely limit their use. Recently, structure activity analysis revealed that the initial pungent and general excitatory effects can be prevented by structural modifications in such a way that the analgesic activity is retained. In this paper we present SDZ 249-665, a capsaicin analogue which produced analgesia in the mouse and anti-hyperalgesic effects in the rat and guinea pig. SDZ 249-665 was administered p.o., s.c. and i.v. in models of nociceptive pain, such as tail flick latency in response to a noxious thermal stimulus and acetic acid-induced writhing in mice, and in models of inflammatory mechanical hyperalgesia induced by turpentine or carrageenan in the rat and guinea pig, respectively. SDZ 249-665 was effective in the tail flick and the writhing assays and produced significant anti-hyperalgesic effects in the inflammatory models. The efficacy of SDZ 245-665 was similar to that of capsaicin, however, it was significantly more potent. SDZ 249-665 did not produce any irritancy in a nose wipe assay in guinea pigs or an eye irritancy assay in rats, while capsaicin was clearly irritant in both cases. Furthermore, unlike capsaicin, SDZ 249-665 did not produce unwanted side effects such as bronchoconstriction and blood pressure changes in the analgesic/anti-hyperalgesic dose range. Thus, a clear analgesic therapeutic window exists for SDZ 249-665. In summary, SDZ 249-665 is a potent orally active, analgesic/anti-hyperalgesic agent in mouse, rat and guinea pig. It lacks the excitatory effects associated with capsaicin and other close analogues, and therefore provides a clear therapeutic window for use in painful conditions. In addition to this favourable profile, no sign of tolerance was detected after a 5 day repeated dose treatment.

Only a few host cell proteins that associate with arenaviruses have been identified. To date, the arenavirus Z protein associates with the promyelocytic leukemia protein PML and the ribosomal P proteins. The majority of PML is present in nuclear bodies which are translocated to the cytoplasm by infection with the arenavirus, lymphocytic choriomeningitis virus (LCMV). The Z protein is a small zinc-binding RING protein with an unknown function which is required for the viral life cycle. Here, we demonstrate an association between Z and the host cell translation factor, eukaryotic initiation factor 4E (eIF-4E) in infected and transfected cells. Z's association with both ribosomal proteins and this translation factor led us to investigate whether Z could modulate host cell translation. In cell culture, Z selectively represses protein production in an eIF-4E-dependent manner. Specifically, we see reduction in cyclin D1 protein production with no effect on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cells transfected with Z. Previous reports indicate that cyclin D1 is sensitive to eIF-4E levels, whereas GAPDH is not. Consistent with this, we observe preferential downregulation of cyclin D1 during infection and no effect on GAPDH. Further, no changes in RNA levels were observed for cyclin D1 or GAPDH transcripts. The interaction between eIF-4E and Z may provide a mechanism for slower growth observed in infected cells and a viral strategy for establishing chronic infection.

Thrombomodulin (TM) is a cofactor for protein C activation by thrombin and each residue of a consensus Ca2+ site in the sixth epidermal growth factor domain (EGF6) is essential for this cofactor activity [Nagashima, M., Lundh, E., Leonard, J.C., Morser, J.Parkinson, J.F. (1993) J. Biol. Chem. 268, 2888-2892]. Three soluble analogs of the extracellular domain of TM, solulin (Glu4-Pro490), TME1-6 (Cys227-Cys462) and TMEi4-6 (Val345-Cys462) were prepared for equilibrium dialysis experiments by exhaustive dialysis against Ca2+-depleted buffer. However, all three analogs still contained one tightly bound Ca2+ (Kd approximately 2 microm), which could only be removed by EDTA. Epitope mapping with Ca2+-dependent monoclonal antibodies to EGF6 provided further localization of this tight Ca2+ site. Equilibrium dialysis of the soluble TM analogs in [45Ca2+] between 10 and 200 microm revealed a second Ca2+ site (Kd = 30 +/- 10 microm) in both solulin and TME1-6, but not in TMEi4-6. Ca2+ binding to this second site was unaffected by bound thrombin and we attribute it to the consensus Ca2+ site in EGF3. A 75-fold decrease in the binding affinity of thrombin to TM was observed with immobilized solulin treated with EDTA to remove the high affinity Ca2+ by measuring kassoc and kdiss rates in a BIAcoretrade mark instrument. Ca2+-dependent conformational transitions detected by CD spectroscopy in the far UV indicate a more ordered structure upon Ca2+ binding. Bound Ca2+ stabilized soluble TM against protease digestion at a trypsin-like protease-sensitive site between Arg456 and His457 in EGF6 compared with protease treatment in EDTA. Finally, TM containing EGF domains 4-6, but lacking the interdomain loop between EGF3 and 4 (TME4-6), has an identical Ca2+ dependence for the activation of protein C as found for TMEi4-6, indicating this interdomain loop is not involved in Ca2+ binding.

Neuropathic pain is poorly managed by conventional analgesic therapy, such as non-steroidal anti-inflammatory drugs and opiates. The development of animal models of peripheral neural damage has aided in our understanding of the pathology and pharmacology of neuropathic pain. This report is the first clear demonstration using selective neurokinin-1 receptor antagonists of a potentially novel therapeutic approach to the treatment of neuropathic pain resulting from peripheral nerve damage in a guinea-pig model. The neurokinin-1 receptor antagonists, SDZ NKT 343 and LY 303,870 significantly reduced mechanical hyperalgesia following oral and intrathecal administration. (R,R)-SDZ NK T343, the enantiomer of SDZ NKT 343 did not show anti-hyperalgesic activity. RPR 100,893 showed significant anti-hyperalgesic activity only following intrathecal administration suggesting poor absorption or low level penetration of the blood-brain barrier. These results imply that neurokinin-1 receptor antagonists offer a new class of anti-hyperalgesic drugs with a largely central site of action in neuropathic pain.

A lead compound which had sub-micromolar affinity for the rabbit NK1 receptor but negligible affinity for rat NK1 receptors, 3a, was discovered by directed screening. 2-Substitution in the ring of the benzylthiourea substituent in the initial lead was found to be important, and halogens (Cl, Br) in this position were found to improve affinity for the human receptor. The activity of a series of 2-halo-substituted benzylthioureas was then optimized by modification of the proline diphenylmethyl amide, guided by a simple conceptual model based on structural overlay between these early antagonists and NK1 selective peptides. In this way, aromatic amino acid amides were identified which had improved affinity with respect to the starting diphenylmethyl (DPM) amides. The first sub-nanomolar ligand for the human NK1 receptor which arose from this series, 4af, combined a 2-chlorobenzylthiourea unit with a 2-naphthylalanine amide. Contemporaneously it was discovered that the benzylthiourea unit could be simplified to a phenylthiourea providing that an appropriate 2-substituent was also incorporated. Combination of these two series gave 2-NO2 phenylthiourea analogues which led directly to the analogous urea, 5f (2-nitrophenylcarbamoyl-(S)-prolyl-(S)-3-(2-naphthyl)alanyl-N-benz yl- N-methylamide, SDZ NKT 343), a highly potent ligand for the human NK1 receptor (Ki = 0.16 nM). In addition to its high in vitro potency, 5f proved to be a potent orally active analgesic in guinea pig models of chronic inflammatory and neuropathic pain. The nature of the 2-aryl substituent was found to be critical for oral activity in this series. Clinical evaluation of 5f as a novel analgesic agent is currently underway.

During the development of purification procedures for Escherichia coli RNA polymerase (RNAP), we noticed the consistent co-purification of a 110-kDa polypeptide. Here, we report the identification of the 110-kDa protein as the product of the hepA gene, a member of the SNF2 family of putative helicases. We have cloned the hepA gene and overexpressed and purified the HepA protein. We show in vitro that RNAP preparations have an ATPase activity only in the presence of HepA and that HepA binds core RNAP competitively with the promoter specificity sigma70 subunit with a 1:1 stoichiometry and a dissociation constant (Kd) of 75 nM. An E. coli strain with a disruption in the hepA gene shows sensitivity to ultraviolet light.