Message Level Research Articles

Investigation of the Reciprocal Relationship between the Expression of Two Gap Junction Connexin Proteins, Connexin46 and Connexin43

Connexins are the transmembrane proteins that form gap junctions between adjacent cells. The function of the diverse connexin molecules is related to their tissue-specific expression and highly dynamic turnover. Although multiple connexins have been previously reported to compensate for each other's functions, little is known about how connexins influence their own expression or intracellular regulation. Of the three vertebrate lens connexins, two connexins, connexin43 (Cx43) and connexin46 (Cx46), show reciprocal expression and subsequent function in the lens and in lens cell culture. In this study, we investigate the reciprocal relationship between the expression of Cx43 and Cx46. Forced depletion of Cx43, by tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate, is associated with an up-regulation of Cx46 at both the protein and message level in human lens epithelial cells. An siRNA-mediated down-regulation of Cx43 results in an increase in the level of Cx46 protein, suggesting endogenous Cx43 is involved in the regulation of endogenous Cx46 turnover. Overexpression of Cx46, in turn, induces the depletion of Cx43 in rabbit lens epithelial cells. Cx46-induced Cx43 degradation is likely mediated by the ubiquitin-proteasome pathway, as (i) treatment with proteasome inhibitors restores the Cx43 protein level and (ii) there is an increase in Cx43 ubiquitin conjugation in Cx46-overexpressing cells. We also present data that shows that the C-terminal intracellular tail domain of Cx46 is essential to induce degradation of Cx43. Therefore, our study shows that Cx43 and Cx46 have novel functions in regulating each other's expression and turnover in a reciprocal manner in addition to their conventional roles as gap junction proteins in lens cells.

Abstract 4600: Green tea polyphenols causes cell cycle arrest and apoptosis of prostate cancer cells by suppressing class I histone deacetylases

Abstract Green Tea polyphenols (GTP) possess anticancer activity and causes cell cycle arrest and apoptosis in a majority of cancer cells however the mechanism(s) involved in this regulation is still unclear. Recent studies from our group [Int J Cancer 126:2520-33, 2010] reported that GTP has ability to alter gene expression by epigenetic modifications including acetylation/deacetylation of histones. Histone acetyltranferases (HATs) and deacetylases (HDACs) enzymes play important role in these modifications. Acetylation relaxes chromatin, making it accessible to transcription factors and increased gene expression. Deacetylation, on the contrary, represses gene expression. HDACs are grouped into 4 classes: class I (HDACs1-3 & 8), class II (HDACs 4-7 and HDACs 9-10), class III (Sirt1-Sirt7) and class IV (HDAC11). Class I HDACs are overexpressed in many cancer types including prostate cancer and their inhibition leads to cell cycle arrest and apoptosis. Since many dietary agents have been reported to cause cell cycle arrest and apoptosis in cancer cells by inhibiting class I HDACs, we propose similar mechanism of action by GTP. Androgen-responsive human prostate cancer LNCaP (harboring WT p53) and androgen-refractory PC-3 (lacking p53) cells were treated with different doses of GTP (10-80µg/ml) for 24h and expression of HDAC1, 2, 3 and 8, p53, p21, Bax and Bcl2 at protein and message levels were determined. Treatment with GTP resulted in dose-dependent decrease in the expression of HDAC 2, 3 and 8 in both prostate cancer cells, but not in HDAC 1, however total HDAC activity was decreased at the highest dose of 80µg/ml GTP. Subsequently, increased acetylation of histone H3 at Lys 9/18 and Histone H4 was observed after GTP treatment. GTP treatment resulted in increased expression of p21and Bax and decrease in Bcl2 at protein and message level in these cells. These alterations were consistent with the G1 phase arrest in dose- and time-dependent manner (69%-80% in LNCaP cells and 40%-62% in PC-3 cells) after GTP treatment. Exposure of cells with GTP up to 72 h resulted in the induction of apoptosis in both cell lines (2.7-84.5% in LNCaP cells and 4.0-85.2% in PC3 cells) as measured by annexin V assay. Furthermore, treatment of cells with proteasomal inhibitor, MG132 together with GTP prevented degradation of class I HDACs, compared to cells treated with GTP alone, indicating increased proteasomal degradation of class I HDACs by GTP. Taken together, we conclude that inhibition of class I HDACs and increased acetylation of histone proteins by GTP contributes to the mechanism of GTP-induced cell cycle arrest and apoptosis of prostate cancer cells, irrespective of their p53 status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4600. doi:10.1158/1538-7445.AM2011-4600

Collagen I Induces Discoidin Domain Receptor (DDR) 1 Expression through DDR2 and a JAK2-ERK1/2-mediated Mechanism in Primary Human Lung Fibroblasts

Discoidin domain receptors (DDRs) DDR1 and DDR2 are receptor tyrosine kinases with the unique ability among receptor tyrosine kinases to respond to collagen. Several signaling molecules have been implicated in DDR signaling, including Shp-2, Src, and MAPK pathways, but a detailed understanding of these pathways and their transcriptional targets is still lacking. Similarly, the regulation of the expression of DDRs is poorly characterized with only a few inflammatory mediators, such as lipopolysaccharide and interleukin-1β identified as playing a role in DDR1 expression. DDRs have been reported to induce the expression of various genes including matrix metalloproteinases and bone morphogenetic proteins, but the regulatory mechanisms underlying DDR-induced gene expression remain to be determined. The aim of the present work was to elucidate the molecular mechanisms implicated in the expression of DDRs and to identify DDR-induced signaling pathways and target genes. Our data show that collagen I induces the expression of DDR1 in a dose- and time-dependent manner in primary human lung fibroblasts. Furthermore, activation of DDR2, JAK2, and ERK1/2 MAPK signaling pathways was essential for collagen I-induced DDR1 and matrix metalloproteinase 10 expression. Finally, inhibition of the ERK1/2 pathway abrogated DDR1 expression by blocking the recruitment of the transcription factor polyoma enhancer A-binding protein 3 to the DDR1 promoter. Our data provide new insights into the molecular mechanisms of collagen I-induced DDR1 expression and demonstrate an important role for ERK1/2 activation and the recruitment of polyoma enhancer-A binding protein 3 to the DDR1 promoter.

RETRACTED: Pharmacologic Blockade of 5-Lipoxygenase Improves the Amyloidotic Phenotype of an Alzheimer's Disease Transgenic Mouse Model: Involvement of γ-Secretase

The 5-lipoxygenase (5-LO) enzyme is widely distributed within the central nervous system. Previous works showed that this protein is up-regulated in Alzheimer's disease (AD) and that its genetic absence results in a reduction of amyloid β (Aβ) levels in Tg2576 mice. In the present study, we examined the effect of 5-LO pharmacological inhibition on the amyloidotic phenotype of these mice. Aβ deposition in the brains of mice receiving zileuton, a selective and specific 5-LO inhibitor, was significantly reduced when compared with control Tg2576 mice receiving vehicle. This reduction was associated with a similar decrease in brain Aβ peptides levels. Zileuton treatment did not induce any change in the steady state levels of amyloid-β precursor protein (APP), BACE1 or ADAM10. By contrast, it resulted in a significant reduction of presenilin 1 (PSEN1, alias PS1), nicastrin (NCSTN) , presenilin enhancer 2 homolog (PSNEN, alias, Pen-2), and anterior pharynx defective 1 (APH-1), the four components of the γ-secretase complex-at the protein and message level. Furthermore, in vitro studies confirmed that zileuton prevents Aβ formation by modulating γ-secretase complex levels without affecting Notch signaling. These data establish a functional role for 5-LO in the pathogenesis of AD-like amyloidosis, whereby it modulates the γ-secretase pathway. They suggest that pharmacological inhibition of 5-LO could provide a novel therapeutic opportunity for AD.

Safeguarding reputation through strategic, integrated and situational crisis communication management

PurposeThe purpose of this paper is to address the often missing theoretical foundation of crisis communication from an integrated perspective on the micro, meso and macro level. Based on the theory of structuration, a systematic, integrative framework is developed for safeguarding organizational legitimization and multidimensional reputation through communication during crisis situations which is applicable both for profit and non‐profit organizations.Design/methodology/approachGidden's theory of structuration was chosen as a basis to develop the integrative model of crisis communication that proposes a communicative impact on reputation on a situative level of message strategies (micro level), an organizational level (meso level) and a societal level (macro level). A well‐organized crisis communication management on all of these levels is seen as the key communicative driver to safeguard long‐term organizational reputation.FindingsThe paper shows that successful crisis communication management must be conceptualized and addressed on distinctive levels of complexity. While on a message level (situative crisis communication) it creates meaning, crisis communication must be seen as management task on an organizational level (integrative crisis communication). However, in order to fully safeguard reputation in the long term and trustworthiness in the short term, crisis communication has also a societal component when addressing moral standards and norms (strategic crisis communication).Research limitations/implicationsThe paper is a conceptual contribution which build the basis of a follow‐up empirical, experimental study where the proposed model is successfully tested.Practical implicationsFor PR managers, this paper gives reasons to conceptualize crisis communication management, not only on a message strategy level, but also to take into consideration the organizational and societal levels.Originality/valueThe paper stands in line with the theoretical discourse of organizational crisis communication. So far, few approaches conceptualize organizational crisis communication thoroughly on an integrated level of different perspectives so that the paper provides an important input, pushing the discussion forward.

Defending Against XML-Based Attacks Using State-Based XML Firewall

With the proliferation of service-oriented systems and cloud computing, web services security has gained much attention in recent years. Web service attacks, called XML-based attacks, typically occur at the SOAP message level, thus they are not readily handled by existing security mechanisms such as a conventional firewall. In order to provide effective security mechanisms for service-oriented systems, XML firewalls have recently been introduced as one of the major means for web services security. In this paper, we present a framework for state-based XML firewall, called S-Wall, which supports dynamic role-based access control (D-RBAC) and detection of XML-based attacks in real-time. We provide a detailed design of the S-Wall security model by defining state-based information, user information, access control policies, and detection and verification (D&V) rules. The D&V rules are modularized into separate units, which support real-time detection and verification of various types of attacks using state-based information. To illustrate the effectiveness of our approach, we develop a prototype S-Wall, and utilize a case study to demonstrate how S-Wall can be used to efficiently detect and defend against XML-based attacks.

SAWSDL Service Discovery Based on Fine-Grained Data Semantics

With the aim to improve the effectiveness of SAWSDL service discovery, this paper proposes a novel discovery method for SAWSDL services, which is based on the matchmaking of so-called fine-grained data semantics that is defined via sets of atomic elements with built-in data types. The fine-grained data semantics can be obtained by a transformation algorithm that decomposes parameters at message level into a set of atomic elements, considering the characteristics of SAWSDL service structure and semantic annotations. Then, a matchmaking algorithm is proposed for the matching of fine-grained data semantics, which avoids the complex and expensive structural matching at the message level. The fine-grained data semantics is transparent to the specific data structure of message-level parameters, therefore, it can help to match successfully similar Web services with different data structures of parameters. Moreover, a comprehensive measure is proposed by considering together several important components of SAWSDL service descriptions at the same time. Finally, this method is evaluated on SAWSDL service discovery test collection SAWSDL-TC2 and compared with other SAWSDL matchmakers. The experimental results show that our method can improve the effectiveness of SAWSDL service discovery with low average query response time. The results imply that fine-grained parameters fit to represent the data semantics of SAWSDL services, especially when data structures of parameters are not important for semantics.

The WNKs: Atypical Protein Kinases With Pleiotropic Actions

WNKs are serine/threonine kinases that comprise a unique branch of the kinome. They are so-named owing to the unusual placement of an essential catalytic lysine. WNKs have now been identified in diverse organisms. In humans and other mammals, four genes encode WNKs. WNKs are widely expressed at the message level, although data on protein expression is more limited. Soon after the WNKs were identified, mutations in genes encoding WNK1 and -4 were determined to cause the human disease familial hyperkalemic hypertension (also known as pseudohypoaldosteronism II, or Gordon's Syndrome). For this reason, a major focus of investigation has been to dissect the role of WNK kinases in renal regulation of ion transport. More recently, a different mutation in WNK1 was identified as the cause of hereditary sensory and autonomic neuropathy type II, an early-onset autosomal disease of peripheral sensory nerves. Thus the WNKs represent an important family of potential targets for the treatment of human disease, and further elucidation of their physiological actions outside of the kidney and brain is necessary. In this review, we describe the gene structure and mechanisms regulating expression and activity of the WNKs. Subsequently, we outline substrates and targets of WNKs as well as effects of WNKs on cellular physiology, both in the kidney and elsewhere. Next, consequences of these effects on integrated physiological function are outlined. Finally, we discuss the known and putative pathophysiological relevance of the WNKs.

Epigenetic Silencing of Mir-218 Regulates CDK6 Expression In Pre-B Acute Lymphoblastic Leukemia Cells

AbstractAbstract 3621Increasing evidence suggests that dysregulation of miRNAs plays an important pathological role in various malignant diseases including acute leukemia. To reveal the contributions of aberrant epigenetic modifications to the deregulated miRNA expression in precursor B-cell acute lymphoblastic leukemia, we examined the miRNA expression profile in NALM-6 cells after treatment with the combination of 5-AZA-2'-deoxycytidine (AZA) and trichostatin A (TSA). We found that the treatment significantly increased expression of 34 miRNAs and decreased the expression of 10 miRNAs. One of the most significantly upregulated miRNAs is miR-218, an intronic miRNA that can be transcribed from either pri-miR-218-1 or pri-miR-218-2, residing in the intron of the SLIT2 gene or SLIT3 gene respectively. Interestingly, we detected that pri-miR-218-1 and its host gene SLIT2, but not pri-miR-218-2 and SLIT3, were induced by AZA plus TSA treatment. Consistent with this observation, we showed that the CpG islands in SLIT2 promoter was highly methylated in NALM-6 cells and AZA plus TSA treatment significantly decreased DNA methylaiton in this region. We found that targeting of the 3'untranslated region of CDK6, a bona fide oncogenic factor, by miR-218 resulting in translational repression. Overexpression of miR-218 expression in NALM-6 cells by transfection of miR-218 precursor decreased cellular expression of CDK6 at the protein level, but not at the message level. AZA and TSA treatment decreased CDK6 expression in NALM-6 cells, presumably through upregulating miR-218. Our results indicate that epigenetic regulation plays an important role in controlling miRNA expression in human acute lymphoblastic leukemia cells. Epigenetic silencing of miR-218 may contribute to the overexpression of CDK6 in NALM-6 cells. Disclosures:No relevant conflicts of interest to declare.

Proteomic comparison of spherical aggregates and adherent cells of cardiac stem cells

Background Previously published papers showed that cardiac stem cells (CSCs) form (cardio)sphere. However, recent studies questioned the significance of the sphere-formation as one of the characteristics of CSCs. We isolated c-kit-positive cardiac stem cells, cultured as bulk (CSC-BC) and characterized them previously. Among them, CSC-BC21 formed an extraordinary number of spheres. Using a clone derived from this bulk culture, we investigated the effect of sphere-formation on differentiation and performed proteomics analysis comparing two statuses, cardiosphere and dish substrate attachment. Methods We performed sphere-forming assay to compare the sphere-forming ability among CSC-BCs. The cloned cells from CSC-BC21, which had distinct sphere-forming ability, were cultured in a differentiation medium (DM) to induce cardiac myocyte differentiation. We performed RT-PCR analysis to investigate if cardiosphere-formation affects cardiac myocyte gene expression level. Furthermore, proteome analysis was performed to compare floating cardiosphere (flCS) and dish-attached cardiosphere-derived cells (daCS). Results One of the cloned cells, CSC-21E expressed higher troponin I message than CSC-BC21. Moreover, the message level of troponin I was enhanced when they had experienced cardiosphere prior to the treatment of myocyte differentiation medium. The change from flCS to daCS accompanied up-regulation of chaperones and down regulation of glycolytic and other metabolic enzymes. Calreticulin and Hsp 90 were among the up-regulated chaperons. Calreticulin is known to be an essential component of cardiogenesis. Conclusion These results suggest that the switch from aggregated sphere to the cell attachment, is important for advancing the cardiac cell differentiation.

Abstract 1439: De novo expression of c-kit in prostate cancer cells interacting with the bone microenvironment

Abstract Specific cellular and molecular interactions between prostate cancer (PC) cells and the bone microenvironment lead to expansion of the metastatic deposit, disease progression, and ultimately significant morbidity and mortality. We recently reported an association between expression of the tyrosine kinase receptor c-kit and clinical PC progression, with the highest expression seen in clinical metastatic bone specimens. Moreover, we found that intraosseous tumors formed by otherwise c-kit negative PC3 cells injected into human bone xenografts in SCID mice (SCID-hu model) expressed c-kit. To determine the relevance of these findings, we examined additional human PC cell lines, namely LNCaP, C42b, and DU145, for the expression of c-kit in vitro and in vivo. While none of the cultured PC cells expressed c-kit at the message or protein level, immunostaining for c-kit in intraosseous tumors produced by these cells in the SCID-hu model revealed a distinctive and specific reactivity in the cancer cell compartment. The induction of c-kit expression in PC cells growing within the bone microenvironment was emulated by co-culture of PC3 cells and bone-derived cells embedded in Matrigel, as demonstrated by RT-PCR analysis. In an attempt to mimic the de novo c-kit expression observed in the SCID-hu model and to identify potential signaling pathways triggered by c-kit activation by its ligand stem cell factor (SCF), normally expressed by the bone microenvironment, we transiently transfected PC3 cells with wild-type c-kit. Activation of c-kit was confirmed by immunoblotting for phosphorylated c-kit. Using the whole human genome Agilent microarray, we found that 220 genes were differentially expressed when SCF-treated and non-treated c-kit-transfected PC3 cells were compared, resulting in discrete functional sub-networks that were coordinately up or down-regulated. Most notably, pathway and genetic network analysis revealed that c-kit activation resulted in an up-regulated fibroblast growth factor-2-mediated sub-network. Taken together, these results suggest that c-kit induction in PC cells in the bone microenvironment modulates cell-cell interaction pathways and growth factor signaling systems that ultimately lead to expansion of bone metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1439.

The BCL-2 Protein BAK Is Required for Long-chain Ceramide Generation during Apoptosis

The BCL-2 family members BAK and BAX are required for apoptosis and trigger mitochondrial outer membrane permeabilization (MOMP). Here we identify a MOMP-independent function of BAK as a required factor for long-chain ceramide production in response to pro-apoptotic stress. UV-C irradiation of wild-type (WT) cells increased long-chain ceramides; blocking ceramide generation prevented caspase activation and cell death, demonstrating that long-chain ceramides play a key role in UV-C-induced apoptosis. In contrast, UV-C irradiation did not increase long-chain ceramides in BAK and BAX double knock-out cells. Notably, this was not specific to the cell type (baby mouse kidney cells, hematopoietic) nor the apoptotic stimulus employed (UV-C, cisplatin, and growth factor withdrawal). Importantly, long-chain ceramide generation was dependent on the presence of BAK, but not BAX. However, ceramide generation was independent of the known downstream actions of BAK in apoptosis (MOMP or caspase activation), suggesting a novel role for BAK in apoptosis. Finally, enzymatic assays identified ceramide synthase as the mechanism by which BAK regulates ceramide metabolism. There was no change in CerS expression at the message or protein level, indicating regulation at the post-translational level. Moreover, CerS activity in BAK KO microsomes can be reactivated upon addition of BAK-containing microsomes. The data presented indicate that ceramide-induced apoptosis is dependent upon BAK and identify a novel role for BAK during apoptosis. By establishing a unique role for BAK in long-chain ceramide metabolism, these studies further demonstrate that the seemingly redundant proteins BAK and BAX have distinct mechanisms of action during apoptosis induction.

Identification of MyD88 as a novel target of miR-155, involved in negative regulation of Helicobacter pylori-induced inflammation

MicroRNA-155 (miR-155) has been implicated as a central regulator of the immune system. We have previously reported that miR-155 negatively regulates Helicobacter pylori ( H. pylori)-induced inflammation, but the molecular mechanism of miR-155 regulating the inflammation is not fully clear. Here, we identified myeloid differentiation protein 88 (MyD88) as a target gene of miR-155, and found that miR-155 decreased MyD88 expression at the protein but not the mRNA message level, suggesting that the miR-155-mediated inhibition is a post-transcriptional event. Furthermore, the overexpression of miR-155 led to significantly reduced IL-8 production induced by H. pylori infection. Thus, we have demonstrated that miR-155 can negatively regulate inflammation by targeting a key adaptor molecule MyD88 in inflammatory pathways.

Realization of Threats and Countermeasure in Semantic Web Services

Semantic Web services (SWS) are worn by large number of companies as they rendering products and services to customers and business partners throughout the Internet and corporate extranets. The security necessities for these service providers are of supreme importance. Platform-based security services are provided by operating system. Internet Information Services can be used to offer point-to-point business solutions. However message based architecture of SWS is increasingly being used, so new challenges automatically inherited from previous one. All these scenarios require security to be deal with at the message level, transmission level and mutual authentication to carry out cross-platform interoperability and routing through multiple middle level agent nodes. Semantic Web service environment (SWSE) need to implement different level security solutions for including authentication, identification, privacy and data integrity. The threats of network eavesdropping or information disclosure at intermediate application nodes must be addressed if our SWS requests or responses messages convey sensitive application data as credit card numbers, SSN number, employee details, and so on. We will discuss various categories of vulnerabilities and find out causes of threats. Countermeasures will be part of our discussion like Symmetric encryption using shared keys, asymmetric encryption using X.509 certificates, and Digital signature.

Identification of a Novel Family of Laminin N-terminal Alternate Splice Isoforms

The laminins are a family of heterotrimeric basement membrane proteins that play roles in cellular adhesion, migration, and tissue morphogenesis. Through in silico analysis of the laminin-encoding genes, we identified a novel family of alternate splice isoforms derived from the 5′-end of the LAMA3 and LAMA5 genes. These isoforms resemble the netrins in that they contain a laminin N-terminal domain followed by a short stretch of laminin-type epidermal growth factor-like repeats. We suggest the terms LaNt (laminin N terminus) α3 and LaNt α5, for the predicted protein products of these mRNAs. RT-PCR confirmed the presence of these transcripts at the mRNA level. Moreover, they exhibit differential, tissue-specific, expression profiles. To confirm the existence of LaNt α3 protein, we generated an antibody to a unique domain within the putative polypeptide. This antibody recognizes a protein at the predicted molecular mass of 64 kDa by immunoblotting. Furthermore, immunofluorescence analyses revealed a basement membrane staining in epithelial tissue for LaNt α3 and LaNt α3 localized along the substratum-associated surface of cultured keratinocytes. We have also tested the functionality LaNt α3 through RNAi-mediated knockdown. Keratinocytes exhibiting specific knockdown of LaNt α3 displayed impaired adhesion, stress resistance, and reduced ability to close scratch wounds in vitro.

The role of dendritic cell subsets in 2,4,6-trinitrobenzene sulfonic acid-induced ileitis

Dendritic cells (DCs) are widely distributed throughout the lymphoid and nonlymphoid tissues, and are important initiators of acquired immunity and also serve as regulators by inducing self-tolerance. However, it has not been thoroughly clarified whether DCs are involved in the termination of immune responses. In this paper, we have examined the kinetical movement of dendritic cells (DCs) in the lamina propria using the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced ileitis model (an animal model for Crohn's disease). Increased numbers of DCs were recruited to the inflammatory sites from day 1 to day 3 at which time the inflammatory responses was clearly observed, then gradually decreased to a steady-state level on day 7 along with the cessation of responses. Three subsets of DCs, PIR-A/B high, PIR-A/B med, and PIR-A/B − DCs in the CD11c +/B220 − conventional DCs (cDCs) were noted on day 3; the number of PIR-A/B med cDCs increased when the inflammatory responses ceased on day 7. The expression of costimulatory molecules such as CD86 and CD54 was lower in the PIR-A/B med DCs compared with the other two cDC subsets or splenic DCs. Furthermore, the stimulatory activity of PIR-A/B med cDCs was lower than those of PIR-A/B high or PIR-A/B − cDCs, and far lower than that of splenic DCs. In addition, an increase in the message level of IL-10 was clearly observed in the PIR-A/B med cDCs on day 7 while that of proinflammatory cytokines such as IL-6 and IL-12 was low. These data demonstrate that PIR-A/B med cDCs which increase at the final stage of inflammation may be involved in the termination of the TNBS-induced ileitis by the delivery of anergic signals to effector T cells due to the lower expressions of costimulatory molecules and the production of immunoregulatory cytokine.

Continuous ST-Segment Monitoring: 3 Case Studies in Progressive Care

Continuous ST-Segment Monitoring: 3 Case Studies in Progressive Care

Gender Bender: Gender Errors in L2 Pronoun Production

To address questions about information processing at the message level, pronoun errors of second language (L2) speakers of English were studied. Some L2 pronoun errors-he/she confusions by Spanish speakers of L2 English-could be due to differences in the informational requirements of the speakers' two languages, providing a window into the composition of the preverbal message that guides grammatical encoding during language production. To study this, Spanish and French speakers of L2 English were made to answer questions designed to elicit pronouns. Spanish speakers produced significantly more gender errors than any other type of pronoun error, and significantly more gender errors than French speakers. The results are as expected given that Spanish is a pro-drop language where many sentences would not have required gender to be encoded had they been uttered in the L1 instead of the L2. The implications for theories about the preverbal message are discussed.

Does elevated intraocular pressure reduce retinal TRKB-mediated survival signaling in experimental glaucoma?

Reduced retrograde transport of neurotrophins (NT) and their receptors has been hypothesized to contribute directly to retinal ganglion cell (RGC) loss in glaucoma. However, strategies of supplementing NT and NT receptors have failed to avert ultimate RGC death in experimental glaucoma. This study examines the response of major components of the NT system and their interacting proteins in a rat glaucoma model. Unilateral chronic intraocular pressure (IOP) elevation was produced by episcleral vein injection of hypertonic saline ( N = 99). Retinas were collected and grouped by extent of optic nerve injury. Quantitative reverse transcription PCR, western blot analysis and immunohistochemistry were used to determine mRNA and protein levels and protein localization. Out of three RGC-specific Brn3 proteins (Brn3a, b, and c), only Brn3a was significantly downregulated at the message level to 35 ± 4% of fellow values with the severest nerve injury. With IOP elevation, no significant alterations were found in retinal mRNA levels for BDNF, NGF, NT-4/5 or NT-3. The abundance of mature retinal BDNF protein was not significantly affected by elevated IOP, while proBDNF protein decreased linearly with increasing injury grade ( r 2 = 0.50). In retinas with the severest nerve injury, TrkB and TrkC receptor mRNA levels significantly declined to 67 ± 9% and 44 ± 5% of fellow values, respectively. However, the levels of TRKB protein and its phosphorylated form were unchanged. Message level for p75 NTR was linearly upregulated up to 219 ± 26% with increasing injury ( r 2 = 0.46), but no alteration was detected at protein level. The mRNA expression of p75 NTR apoptosis adaptor proteins NADE, NRIF, and Lingo1 were significantly downregulated in retinas with the greatest nerve injury. A positive correlation was found between injury extent and message levels for Jun ( r 2 = 0.23) as well as Junb ( r 2 = 0.27), and RGC labeling of activated JUN protein increased. Atf3 mRNA levels demonstrated a positive linear correlation to the extent of injury ( r 2 = 0.53), resulting in a nearly five-fold increase (482 ± 76%) in eyes with the greatest nerve damage. Among downstream pro-survival signaling components, Erk5 mRNA expression was linearly upregulated ( r 2 = 0.32) up to 157 ± 15% of fellow values in retinas with the severest nerve injury ( p < 0.01). A slight positive correlation was found between NF-κB message levels and injury extent ( r 2 = 0.12). Bcl-xl mRNA levels in the most severely injured retinas were significantly reduced to 83 ± 7% by elevated IOP exposure. Message levels for Erk1/2, Akt1-3 or Bcl2 appeared unaffected. Elevated IOP did not alter mRNA levels of pro-apoptotic Bim, Bax, or p53. This study demonstrates that elevated IOP exposure does not result in a dramatic decrease in retinal levels of either BDNF or its receptor, TrkB. It shows that the responses of NT pathways to elevated IOP are complex, particularly with regard to the role of p75 NTR and Atf3. A better understanding of the roles of these proteins in IOP-induced injury is likely to suggest informed strategies for neuroprotection in glaucoma.

Biochemical and Functional Characterization of Six SIX1 Branchio-oto-renal Syndrome Mutations

Branchio-oto-renal syndrome (BOR) is an autosomal dominant developmental disorder characterized by hearing loss, branchial arch defects, and renal anomalies. Recently, eight mutations in the SIX1 homeobox gene were discovered in BOR patients. To characterize the effect of SIX1 BOR mutations on the EYA-SIX1-DNA complex, we expressed and purified six of the eight mutants in Escherichia coli. We demonstrate that only the most N-terminal mutation in SIX1 (V17E) completely abolishes SIX1-EYA complex formation, whereas all of the other mutants are able to form a stable complex with EYA. We further show that only the V17E mutant fails to localize EYA to the nucleus and cannot be stabilized by EYA in the cell. The remaining five SIX1 mutants are instead all deficient in DNA binding. In contrast, V17E alone has a DNA binding affinity similar to that of wild type SIX1 in complex with the EYA co-factor. Finally, we show that all SIX1 BOR mutants are defective in transcriptional activation using luciferase reporter assays. Taken together, our experiments demonstrate that the SIX1 BOR mutations contribute to the pathology of the disease through at least two different mechanisms that involve: 1) abolishing the formation of the SIX1-EYA complex or 2) diminishing the ability of SIX1 to bind DNA. Furthermore, our data demonstrate for the first time that EYA: 1) requires the N-terminal region of the SIX1 Six domain for its interaction, 2) increases the level of the SIX1 protein within the cell, and 3) increases the DNA binding affinity of SIX1.