Cell-specific regulation of acetylcholinesterase expression under inflammatory conditions

Acetylcholine (ACh) has been shown to exert an anti-inflammatory function by down-modulating the expression of pro-inflammatory cytokines. Its availability can be regulated at different levels, namely at its synthesis and degradation steps. Accordingly, the expression of acetylcholinesterase (AChE), the enzyme responsible for ACh hydrolysis, has been observed to be modulated in inflammation. To further address the mechanisms underlying this effect, we aimed here at characterizing AChE expression in distinct cellular types pivotal to the inflammatory response. This study was performed in the human acute leukaemia monocytyc cell line, THP-1, in human monocyte-derived primary macrophages and in human umbilical cord vein endothelial cells (HUVEC). In order to subject these cells to inflammatory conditions, THP-1 and macrophage were treated with lipopolysaccharide (LPS) from E.coli and HUVEC were stimulated with the tumour necrosis factor α (TNF-α). Our results showed that although AChE expression was generally up-regulated at the mRNA level under inflammatory conditions, distinct AChE protein expression profiles were aurprisingly observed among the distinct cellular types studied. Altogether, these results argue for the existence of cell specific mechanisms that regulate the expression of acetylcholinesterase in inflammation.

Identity – towards a conceptualization

The development of economical relations increases the interaction between organizations and stakeholders. It is no more acceptable to manage an organization under a transactional perspective where suppliers had a fundamental role. Nowadays organizations are being managed under a relational perspective where relations and relationship management in general have consequences in identity management (Hakansson e Snehota, 1989, 1995). A correct perception and management of identity is necessary to achieve distinctiveness in the competitive environment. This way, identity is influenced by relations with stakeholders in general and particularly with competitors. The ICIG concept states that identity is related with the values of the organization and it helps creating distinctiveness in the competitive environment (Van Riel, Balmer, 1997); Baker and Balmer (1997) state that identity is what the organization is; Suvatjis and de Chernatony (2005) refer that expressing identity is a dynamic process that evolves the use of a management model to face context changes; Kapferer (1991) states that brand identity is the project, the self conception of the brand. After reviewing and confronting literature under the plethora of identities’ concepts and perspectives (He, Balmer, 2007) one can’t find an integrative answer with all the elements that contribute to identity of organizations. The authors are strongly interested to contribute to the elimination of this limitation and to answer to strategic management needs. In a marketing context one can find: - the corporate identity approach that is focused in the distinctive attributes of an organization (Abratt, Balmer, Marwick e Fill, Stuart, Balmer and Gray, Alessandri, Suvatjis and de Chernatony) - the brand identity approach (related with the application of corporate identity studies to brands) - Kapferer, Semprini, Aaker, de Chernatony). Kapferer (1991), one of the most prolific authors in this field was the first author to integrate identity in a brand concept. In his view, identity is an emission concept. This idea is shared also by Aaker (1996). Yet, identity has to be managed in a competitive environment which is constantly changing. After reviewing and confronting literature, authors select concepts that are generally accepted by the investigators in order to design a model to analyze and manage identity: - corporate identity models: personality, image/reputation, culture, philosophy, mission, strategy, structure, communication - some of these concepts derive from identity models and others from identity management models; - brand identity models – Kapferer (1991, 2008) identity prism, witch is basis of literature in this field: culture, physical facet, personality, relationship (between brand and consumer), reflected consumer, consumers` self-concept. After discussion authors decide to include other concept in line with other authors` view: country of origin (Aaker, 1996). A discussion eliminates the twin concepts and the final selection is as follows: personality, image/reputation, culture (including philosophy and mission), strategy, structure, communication, culture, physical facet, relationship (between brand and consumer), reflected consumer, consumers` self-concept and according to authors` reflections “relationships” deriving from competitors` actions in competitive environment. Competitors’ actions and decisions have a stronger influence in the organizations` positioning than any other stakeholder as stated before. This is a work in progress towards a new model in identity analysis and management so an exploratory study will follow inquiring experts on identity in order to evaluate these concepts and correct the theoretical perspectives.

Using an integrative approach to assess brand identity

There is a wide agreement that identity is a multidisciplinary concept. The authors consider this an opportunity do develop a framework to assess identity. In a marketing context, literature reveals two approaches on identity: one focus on corporate identity and the other focus on branding. The aim of this paper is to integrate these two approaches to develop a synthesis framework to assess brand identity. Based on literature on identity the authors found nine components related to brand identity. Those components are described in this paper as well as the relation they have with brand identity. The authors hope that this synthesis approach contributes to a better understanding of the brand identity, and are very encouraging for refining this framework in the future.

Corporate brand identity management – proposal of a new framework

The term “corporate brand” has been widely used in literature since the eighties. According to Balmer (1998) this concept tends to be used as an alternative to the concept of corporate identity. The author argues that the use of branding principles to discuss corporate identity has tended to align the area more closely with marketing. However, the literature on brand management (Aaker, 1991; Kapferer, 1991 and de Chernatony and McDonald, 1992), gives little attention to the corporate brand” (p. 985). Based on the concepts of corporate brand, brand identity and B2B relationship, the authors are interested in eliminating this gap in literature by designing a framework of corporate brand identity management. The aim of this investigation is to investigate the impact of B2B relationships in corporate brand identity management. The methodology used is quantitative analysis of surveys and scale development. The originality of this paper is to investigate the influence of the relationship between brands in corporate brand identity. This investigation is very important to help the decisions of the corporate brand managers and academics. According to literature, namely on corporate brands (Balmer 2002b, Hatch and Schultz, 2001, 2003) and on brand identity (Kapferer, 1991, 2008, Aaker, 1996, de Chernatony, 1999) the authors developed a corporate brand identity management framework considering relationships between brands a context variable with definite impact on identity management as stated by Hakansson and Snehota (1989, 1995). These authors consider that organisations´ identity management is pursued under a relational perspective with impact on identity management. Most researchers on identity and corporate brand emphasise the importance of external influences (Kennedy, 1977; King, 1991; de Chernatony, 1999; Balmer and Gray, 2000; Balmer, 2002a). Those influences concern legislation, concurrence, political issues... and stakeholders’ perceptions and reputations (due to the holistic approach demanded by corporate brands). In this context the authors claim the importance of another influence: B2B relationships. This decision is inspired in sociological studies (Mannheim, 1950; and Tajfel and Turner, 1979) regarding individual identity. These authors claim that individuals form their personality by interacting in the social field. The authors argue that corporate brand identity also develops itself under a relational approach. The relationships selected to pursue this investigation are the ones that are developed by Portuguese universities and investigation centres that cooperate by developing investigation. Those centres are administrative and financially autonomous

Corporate brand identity management in a technological faculty – how brand relationships influence students and companies reputations

Identity is traditionally defined as an emission concept [1]. Yet, some research points out that there are external factors that can influence it [2]; [3]; [4]. This subject is even more relevant if one considers corporate brands. According to Aaker [5] the number, the power and the credibility of corporate associations are bigger in the case of corporate brands. Literature recognizes the influence of relationships between companies in identity management. Yet, given the increasingly important role of corporate brands, it is surprising that to date no attempt to evaluate that influence has been made in the management of corporate brand identity. Also Keller and Lehman [6] highlight relationships and costumer experience as two areas requiring more investigation. In line with this, the authors intend to develop an empirical research in order to evaluate the influence of relationships between brands in the identity of corporate brand from an internal perspective by interviewing internal stakeholders (brand managers and internal clients). This paper is organized by main contents: theoretical background, research methodology, data analysis and conclusions and finally cues to future investigation.

Corporate brand identity in higher education: a relational perspective

Identity is traditionally defined as an emission concept (Kapferer, 2008). Yet, some research points out that there are external factors that that can influence it (Kennedy, 1975; Markwick e Fill, 1997; Balmer e Gray, 2000). This subject is even more interesting if one considers corporate brands. According to Aaker (2004) the number, the power and the credibility of corporate associations are bigger in the case corporate brands. Literature recognizes the influence of relationships between companies in identity management (Hakansson and Snehota, 1989, 1995; Hakansson and Ford, 2002). Yet, given the increasingly important role of corporate brands, it is surprising that to date no attempt to evaluate that influence has been made in corporate brand´s identity management and reputation. Also Keller and Lehman (2006) highlight relationships and costumer experience as two areas requiring more investigation. The authors argue that corporate brand´s identity can be developed under a relational perspective using relationships with other recognised brands in order to generate positive reputations in stakeholders. Based in relationship and corporate brand identity management, a framework is developed to identify how corporate brands select, develop and invest in relationships with other brands. The context of the proposed relationship concept is the services area (Dwyer et al, 1987; Moorman et al, 1992; Rauyruen et al, 2005 and Hennig-Thurau and Klee, 1997). An empirical qualitative research is designed using two reputational technological higher education institutions (two corporate brands) acting in Portuguese public higher education market.

Differential expression of the eukaryotic release factor 3 (eRF3/GSPT1) according to gastric cancer histological types

Background: There are now several lines of evidence to suggest that protein synthesis and translation factors are involved in the regulation of cell proliferation and cancer development. Aims: To investigate gene expression patterns of eukaryotic releasing factor 3 (eRF3) in gastric cancer. Methods: RNA was prepared from 25 gastric tumour biopsies and adjacent non-neoplastic mucosa. Real time TaqMan reverse transcription polymerase chain reaction (RT-PCR) was performed to measure the relative gene expression levels. DNA was isolated from tumour and normal tissues and gene dosage was determined by a quantitative real time PCR using SYBR Green dye. Results: Different histological types of gastric tumours were analysed and nine of the 25 tumours revealed eRF3/GSPT1 overexpression; moreover, eight of the 12 intestinal type carcinomas analysed overexpressed the gene, whereas eRF3/GSPT1 was overexpressed in only one of the 10 diffuse type carcinomas (Kruskal-Wallis Test; p , 0.05). No correlation was found between ploidy and transcript expression levels of eRF3/GSPT1. Overexpression of eRF3/GSPT1 was not associated with increased translation rates because the upregulation of eRF3/GSPT1 did not correlate with increased eRF1 levels. Conclusions: Overexpression of eRF3/GSPT1 in intestinal type gastric tumours may lead to an increase in the translation efficiency of specific oncogenic transcripts. Alternatively, eRF3/GSPT1 may be involved in tumorigenesis as a result of its non-translational roles, namely (dis)regulating the cell cycle, apoptosis, or transcription.

Regulation of antioxidant enzymes gene expression in the yeast Saccharomyces cerevisiae during stationary phase

Gene expression of three antioxidant enzymes, Mn superoxide dismutase (MnSOD), Cu,Zn superoxide dismutase (Cu,ZnSOD), and glutathione reductase (GR) was investigated in stationary phase Saccharomyces cerevisiae during menadione-induced oxidative stress. Both GR and Cu,ZnSOD mRNA steady state levels increased, reaching a plateau at about 90 min exposure to menadione. GR mRNA induction was higher than that of Cu,ZnSOD (about 14-fold and 9-fold after 90 min, respectively). A different pattern of response was obtained for MnSOD mRNA, with a peak at about 15 min (about 8-fold higher) followed by a decrease to a plateau approximately 4-fold higher than the control value. However, these increased mRNA levels did not result in increased protein levels and activities of these enzymes. Furthermore, exposure to menadione decreased MnSOD activity to half its value, indicating that the enzyme is partially inactivated due to oxidative damage. Cu,ZnSOD protein levels were increased 2-fold, but MnSOD protein levels were unchanged after exposure to menadione in the presence of the proteolysis inhibitor phenylmethylsulfonyl fluoride. These results indicate that the rates of Cu,ZnSOD synthesis and proteolysis are increased, while the rates of MnSOD synthesis and proteolysis are unchanged by exposure to menadione. Also, the translational efficiency for both enzymes is probably decreased, since increases in protein levels when proteolysis is inhibited do not reflect the increases in mRNA levels. Our results indicate that oxidative stress modifies MnSOD, Cu,ZnSOD, and GR gene expression in a complex way, not only at the transcription level but also at the post-transcriptional, translational, and post-translational levels.

Besnoitia besnoiti protein disulfide isomerase (BbPDI): molecular characterization, expression and in silico modelling

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a'_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.

Expression of YAP4 in Saccharomyces cerevisiae under osmotic stress

YAP4, a member of the yeast activator protein (YAP) gene family, is induced in response to osmotic shock in the yeast Saccharomyces cerevisiae. The null mutant displays mild and moderate growth sensitivity at 0.4 M and 0.8 M NaCl respectively, a fact that led us to analyse YAP4 mRNA levels in the hog1 (high osmolarity glycerol) mutant. The data obtained show a complete abolition of YAP4 gene expression in this mutant, placing YAP4 under the HOG response pathway. YAP4 overexpression not only suppresses the osmosensitivity phenotype of the yap4 mutant but also relieves that of the hog1 mutant. Induction, under the conditions tested so far, requires the presence of the transcription factor Msn2p, but not of Msn4p, as YAP4 mRNA levels are depleted by at least 75% in the msn2 mutant. This result was further substantiated by the fact that full YAP4 induction requires the two more proximal stress response elements. Furthermore we find that GCY1, encoding a putative glycerol dehydrogenase, GPP2, encoding a NAD-dependent glycerol-3-phosphate phosphatase, and DCS2, a homologue to a decapping enzyme, have decreased mRNA levels in the yap4 -deleted strain. Our data point to a possible, as yet not entirely understood, role of the YAP4 in osmotic stress response.

Differential expression of CDC25 phosphatases splice variants in human breast cancer cells

Background: CDC25 phosphatases control cell cycle progression by activating cyclin dependent kinases. The three CDC25 isoforms encoding genes are submitted to alternative splicing events which generate at least two variants for CDC25A and five for both CDC25B and CDC25C. An over-expression of CDC25 was reported in several types of cancer, including breast cancer, and is often associated with a poor prognosis. Nevertheless, most of the previous studies did not address the expression of CDC25 splice variants. Here, we evaluated CDC25 spliced transcripts expression in anti-cancerous drug-sensitive and resistant breast cancer cell lines in order to identify potential breast cancer biomarkers. Methods: CDC25 splice variants mRNA levels were evaluated by semi-quantitative RT-PCR and by an original real-time RT-PCR assay. Results: CDC25 spliced transcripts are differentially expres-sed in the breast cancer cell lines studied. An up-regulation of CDC25A2 variant and an increase of the CDC25C5/C1 ratio are associated to the multidrug-resistance in VCREMS and DOXOR breast cancer cells, compared to their sensitive counterpart cell line MCF-7. Additionally, CDC25B2 tran-script is exclusively over-expressed in VCREMS resistant cells and could therefore be involved in the development of certain type of drug resistance. Conclusions: CDC25 splice variants could represent interesting potential breast cancer prognostic biomarkers.

Gene expression regulation and lineage evolution: the North and South tale of the hybrid polyploid Squalius alburnoides complex

The evolution of hybrid polyploid vertebrates, their viability and their perpetuation over evolutionary time have always been questions of great interest. However, little is known about the impact of hybridization and polyploidization on the regulatory networks that guarantee the appropriate quantitative and qualitative gene expression programme. The Squalius alburnoides complex of hybrid fish is an attractive system to address these questions, as it includes a wide variety of diploid and polyploid forms, and intricate systems of genetic exchange. Through the study of genome-specific allele expression of seven housekeeping and tissue-specific genes, we found that a gene copy silencing mechanism of dosage compensation exists throughout the distribution range of the complex. Here we show that the allele-specific patterns of silencing vary within the complex, according to the geographical origin and the type of genome involved in the hybridization process. In southern populations, triploids of S. alburnoides show an overall tendency for silencing the allele from the minority genome, while northern population polyploids exhibit preferential biallelic gene expression patterns, irrespective of genomic composition. The present findings further suggest that gene copy silencing and variable expression of specific allele combinations may be important processes in vertebrate polyploid evolution.

Differential expression of GSPT1 GGCn alleles in cancer

The human eukaryotic release factor 3a (eRF3a), encoded by the G1 to S phase transition 1 gene (GSPT1; alias eRF3a), is upregulated in various human cancers. GSPT1 contains a GGCn polymorphism in exon 1, encoding a polyglycine expansion in the N-terminal of the protein. The longer allele, GGC12, was previously shown to be associated to cancer. The GGC12 allele was present in 2.2% of colorectal cancer patients but was absent in Crohn disease patients and in the control group. Real-time quantitative RT-PCR analysis showed that the GGC12 allele was present at up to 10-fold higher transcription levels than the GGC10 allele (P < 0.001). No GSPT1 amplifications were detected, and there was no correlation between the length of the alleles and methylation levels of the CpG sites inside the GGC expansion. Using flow cytometry, we compared the levels of apoptosis and proliferation rates between cell lines with different genotypes, but detected no significant differences. Finally, we used a cytokinesis-block micronucleus assay to evaluate the frequency of micronuclei in the same cell lines. Cell lines with the longer alleles had higher frequencies of micronuclei in binucleated cells, which is probably a result of defects in mitotic spindle formation. Altogether, these findings indicate that GSPT1 should be considered a potential proto-oncogene.

Modulation of translation factor's gene expression by histone deacetylase inhibitors in breast cancer cells

The histone deacetylase inhibitors sodium butyrate (NaBu) and trichostatin A (TSA) exhibit anti-proliferative activity by causing cell cycle arrest and apoptosis. The mechanisms by which NaBu and TSA cause apoptosis and cell cycle arrest are not yet completely clarified, although these agents are known to modulate the expression of several genes including cell-cycle- and apoptosis-related genes. The enzymes involved in the process of translation have important roles in controlling cell growth and apoptosis, and several of these translation factors have been described as having a causal role in the development of cancer. The expression patterns of the translation mechanism, namely of the elongation factors eEF1A1 and eEF1A2, and of the termination factors eRF1 and eRF3, were studied in the breast cancer cell line MCF-7 by real-time quantitative reverse transcription-polymerase chain reaction after a 24-h treatment with NaBu and TSA. NaBu induced inhibition of translation factors' transcription, whereas TSA caused an increase in mRNA levels. Thus, these two agents may modulate the expression of translation factors through different pathways. We propose that the inhibition caused by NaBu may, in part, be responsible for the cell cycle arrest and apoptosis induced by this agent in MCF-7 cells.

Characterization of a polyubiquitin gene in T. thermophila and of ubiquitin gene expression during sexual reproduction and under stress conditions

A 5-unit polyubiquitin gene, TTU3, was isolated from a T. thermophila genomic library and sequenced. This gene presents an extra triplet coding for Phe, a AGAGA motif and a putative HSE element in its 5'-non-coding region. The ubiquitin gene expression in this ciliate was investigated by Northern blot hybridization in conjugating cells or cells under stress conditions. Exponentially growing cells express two ubiquitin mRNAs of 0.75 and 1.8 kb and a new species of 1.4 kb is induced under hyperthermic stress. During sexual reproduction of the cells (conjugation) the 1.8-kb mRNA is still transcribed whereas the steady-state population of the 0.75 mRNA transcripts is strongly diminished. Southern blot analysis suggests that ubiquitin in T. thermophila constitutes a large family of about ten members.