Induction Of Gene Products Research Articles

Evasion of I Interferon-Mediated Innate Immunity by Pseudorabies Virus.

Type I interferon (IFN-I) mediated innate immunity serves as the first line of host defense against viral infection, ranging from IFN-I production upon viral detection, IFN-I triggered signaling pathway that induces antiviral gene transcription the antiviral effects of IFN-I induced gene products. During coevolution, herpesviruses have developed multiple countermeasures to inhibit the various steps involved to evade the IFN response. This mini-review focuses on the strategies used by the alphaherpesvirus Pseudorabies virus (PRV) to antagonize IFN-I mediated innate immunity, with a particular emphasis on the mechanisms inhibiting IFN-I induced gene transcription through the JAK-STAT pathway. The knowledge obtained from PRV enriches the current understanding of the alphaherpesviral immune evasion mechanisms and provides insight into the vaccine development for PRV control.

Von Willebrand Factor Type A domain of hCLCA1 is sufficient for U-937 macrophage activation

The human hCLCA1 gene is a member of the CLCA gene family that has a well-documented role in inflammatory airway diseases. Previously, we demonstrated that secreted hCLCA1 plays a role in regulating the innate immune response by activating airway macrophages. However, the mechanism of this regulation remains unclear. In this present study, recombinant proteins containing different hCLCA1 domains are expressed to determine the specific hCLCA1 domain(s) responsible for macrophage activation. Specifically, hCLCA1 constructs containing the hydrolase domain (HYD), the von Willebrand Factor Type A (VWA) domain, and the fibronectin type III (FN3) domain were heterologously expressed and affinity purified through fast protein liquid chromatography. Circular dichroism spectroscopy revealed that the purified hCLCA1 constructs exhibited secondary structure consistent with folded proteins. The VWA domain clearly demonstrated an ability to activate macrophages, inducing an increase in both IL-1β mRNA and protein expression. This activation was associated with the activation of MAPKs and NF-κB pathways, identifying potential mechanistic pathways by which hCLCA1's VWA domain exerts its signaling effect. Altogether, this work identifies a domain with signaling function within hCLCA1, providing a specific target to one of the most highly induced gene products of airway inflammatory disease.

Induction of antibacterial proteins and peptides in the coprophilous mushroom Coprinopsis cinerea in response to bacteria

Bacteria are the main nutritional competitors of saprophytic fungi during colonization of their ecological niches. This competition involves the mutual secretion of antimicrobials that kill or inhibit the growth of the competitor. Over the last years it has been demonstrated that fungi respond to the presence of bacteria with changes of their transcriptome, but the significance of these changes with respect to competition for nutrients is not clear as functional proof of the antibacterial activity of the induced gene products is often lacking. Here, we report the genome-wide transcriptional response of the coprophilous mushroom Coprinopsis cinerea to the bacteria Bacillus subtilis and Escherichia coli. The genes induced upon co-cultivation with each bacterium were highly overlapping, suggesting that the fungus uses a similar arsenal of effectors against Gram-positive and -negative bacteria. Intriguingly, the induced genes appeare to encode predominantly secreted peptides and proteins with predicted antibacterial activities, which was validated by comparative proteomics of the C. cinerea secretome. Induced members of two putative antibacterial peptide and protein families in C. cinerea, the cysteine-stabilized αβ-defensins (Csαβ-defensins) and the GH24-type lysozymes, were purified, and their antibacterial activity was confirmed. These results provide compelling evidence that fungi are able to recognize the presence of bacteria and respond with the expression of an arsenal of secreted antibacterial peptides and proteins.

The Pathways Involved in TLQP-62 Mediated Biological Functions

VGF is neurosecretory protein, belongs to the extendedgranin family of proteins, originallyrecognized as a nerve growth factor (NGF) inducible gene product. It is selectively synthesized mostly in neuronal and neuroendocrine cells. It has a very simple organization. It has several biologically active peptides, described as VGF-derived peptides including APPG-40, APPG-37, GRPE-37, NERP-1, NERP-2, NAPP-129 (VGF 20), VGF 18, HFHH-51 (VGF 6), HHPD-1, AQEE-30 (Peptide V), LQEQ-19, TLQP-21 and TLQP-62 (VGF 10). Among these peptides TLQP-62 is of great significance because of its several remarkable biological effects like regulation of memory formation, glucose homeostasis and insulin secretion, gene transcription, neurogenesis, etc. Although in light of these outstanding behavioral and physiological roles, very few information are available regarding its mode of action. Still, no receptor has been found for TLQP-62 mediated cascading pathways. However, considering the significant biological function of TLQP-62, this review study was done to summarize the pathways involved in TLQP-62 mediated action, based on the information till now. TLQP-62 activates signal transduction cascades by two mechanisms including opening of the calcium channels and modulating the AMPK, PKC and ERK pathways. These events result in an increase in intracellular Ca2+ concentration which permits the secretion of insulin. TLQP-62 would be a significant therapeutics and drug discovery tool for a wide range of diseases to which the peptide related, if TLQP-62 mediated pathways are known, as a whole.

Temporally distinct transcriptional regulation of myocyte dedifferentiation and Myofiber growth during muscle regeneration

BackgroundTissue regeneration requires a series of steps, beginning with generation of the necessary cell mass, followed by cell migration into damaged area, and ending with differentiation and integration with surrounding tissues. Temporal regulation of these steps lies at the heart of the regenerative process, yet its basis is not well understood. The ability of zebrafish to dedifferentiate mature “post-mitotic” myocytes into proliferating myoblasts that in turn regenerate lost muscle tissue provides an opportunity to probe the molecular mechanisms of regeneration.ResultsFollowing subtotal excision of adult zebrafish lateral rectus muscle, dedifferentiating residual myocytes were collected at two time points prior to cell cycle reentry and compared to uninjured muscles using RNA-seq. Functional annotation (GAGE or K-means clustering followed by GO enrichment) revealed a coordinated response encompassing epigenetic regulation of transcription, RNA processing, and DNA replication and repair, along with protein degradation and translation that would rewire the cellular proteome and metabolome. Selected candidate genes were phenotypically validated in vivo by morpholino knockdown. Rapidly induced gene products, such as the Polycomb group factors Ezh2 and Suz12a, were necessary for both efficient dedifferentiation (i.e. cell reprogramming leading to cell cycle reentry) and complete anatomic regeneration. In contrast, the late activated gene fibronectin was important for efficient anatomic muscle regeneration but not for the early step of myocyte cell cycle reentry.ConclusionsReprogramming of a “post-mitotic” myocyte into a dedifferentiated myoblast requires a complex coordinated effort that reshapes the cellular proteome and rewires metabolic pathways mediated by heritable yet nuanced epigenetic alterations and molecular switches, including transcription factors and non-coding RNAs. Our studies show that temporal regulation of gene expression is programmatically linked to distinct steps in the regeneration process, with immediate early expression driving dedifferentiation and reprogramming, and later expression facilitating anatomical regeneration.

The interferon-induced antiviral protein PML (TRIM19) promotes the restriction and transcriptional silencing of lentiviruses in a context-specific, isoform-specific fashion.

BackgroundThe promyelocytic leukemia (PML) protein, a type I interferon (IFN-I)-induced gene product and a member of the tripartite motif (TRIM) family, modulates the transcriptional activity of viruses belonging to various families. Whether PML has an impact on the replication of HIV-1 has not been fully addressed, but recent studies point to its possible involvement in the restriction of HIV-1 in human cells and in the maintenance of transcriptional latency in human cell lines in which HIV-1 is constitutively repressed. We investigated further the restriction of HIV-1 and a related lentivirus, SIVmac, by PML in murine cells and in a lymphocytic human cell line. In particular, we studied the relevance of PML to IFN-I-mediated inhibition and the role of individual human isoforms.ResultsWe demonstrate that both human PML (hPML) and murine PML (mPML) inhibit the early post-entry stages of the replication of HIV-1 and a related lentivirus, SIVmac. In addition, HIV-1 was transcriptionally silenced by mPML and by hPML isoforms I, II, IV and VI in MEFs. This PML-mediated transcriptional repression was attenuated in presence of the histone deacetylase inhibitor SAHA. In contrast, depletion of PML had no effect on HIV-1 gene expression in a human T cell line. PML was found to contribute to the inhibition of HIV-1 by IFN-I. Specifically, IFN-α and IFN-β treatments of MEFs enhanced the PML-dependent inhibition of HIV-1 early replication stages.ConclusionsWe show that PML can inhibit HIV-1 and other lentiviruses as part of the IFN-I-mediated response. The restriction takes place at two distinct steps, i.e. reverse transcription and transcription, and in an isoform-specific, cellular context-specific fashion. Our results support a model in which PML activates innate immune antilentiviral effectors. These data are relevant to the development of latency reversal-inducing pharmacological agents, since PML was previously proposed as a pharmacological target for such inhibitors. This study also has implications for the development of murine models of HIV-1.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0253-1) contains supplementary material, which is available to authorized users.

Cognitive Improvement by Photic Stimulation in a Mouse Model of Alzheimer's Disease.

We previously reported that activity of the large conductance calcium-activated potassium (big-K, BK) channel is suppressed by intracellular Aβ in cortical pyramidal cells, and that this suppression was reversed by expression of the scaffold protein Homer1a in 3xTg Alzheimer's disease model mice. Homer1a is known to be expressed by physiological photic stimulation (PS) as well. The possibility thus arises that PS also reverses Aβ-induced suppression of BK channels, and thereby improves cognition in 3xTg mice. This possibility was tested here. Chronic application of 6-hour-long PS (frequency, 2 Hz; duty cycle, about 1/10; luminance, 300 lx) daily for 4 weeks improved contextual and tone-dependent fear memory in 3xTg mice and, to a lesser extent, Morris water maze performance as well. Hippocampal long-term potentiation was also enhanced after PS. BK channel activity in cingulate cortex pyramidal cells and lateral amygdalar principal cells, suppressed in 3xTg mice, were facilitated. In parallel, neuronal excitability, elevated in 3xTg mice, was recovered to the control level. Gene expression of BK channel, as well as that of the scaffold protein Homer1a, was found decreased in 3xTg mice and reversed by PS. It is known that Homer1a is an activity-dependently inducible immediate early gene product. Consistently, our previous findings showed that Homer1a induced by electrical stimulation facilitated BK channels. By using Homer1a knockouts, we showed that the present PS-induced BK channel facilitation is mediated by Homer1a expression. We thus propose that PS might be potentially useful as a non-invasive therapeutic measure against Alzheimer's disease.

Tumor-associated macrophages in oral premalignant lesions coexpress CD163 and STAT1 in a Th1-dominated microenvironment

BackgroundTumor-associated macrophages (TAMs) are implicated in the growth, invasion and metastasis of various solid tumors. However, the phenotype of TAMs in premalignant lesions of solid tumors has not been clarified. In the present study, we identify the phenotype of TAMs in leukoplakia, an oral premalignant lesion, by immunohistochemical analysis and investigate the involvement of infiltrated T cells that participate in the polarization of TAMs.MethodsThe subjects included 30 patients with oral leukoplakia and 10 individuals with normal mucosa. Hematoxylin and eosin slides were examined for the histological grades, and immunohistochemical analysis was carried out using antibodies against CD68 (pan-MΦ), CD80 (M1 MΦ), CD163 (M2 MΦ), CD4 (helper T cells: Th), CD8 (cytotoxic T cells), CXCR3, CCR5 (Th1), CCR4 (Th2), signal transducer and activator of transcription (STAT1), phosphorylated STAT1 (pSTAT1) and chemokine CXCL9. The differences in the numbers of positively stained cells among the different histological grades were tested for statistical significance using the Kruskal-Wallis test. Correlations between different types of immune cells were determined using Spearman’s rank analysis.ResultsAn increase in the rate of CD163+ TAM infiltration was observed in mild and moderate epithelial dysplasia, which positively correlated with the rate of intraepithelial CD4+ Th cell infiltration. Although CCR4+ cells rarely infiltrated, CXCR3+ and CCR5+ cells were observed in these lesions. Cells positive for STAT1 and chemokine CXCL9, interferon- (IFN)-induced gene products, and pSTAT1 were also observed in the same lesions. Double immunofluorescence staining demonstrated that the cells that were positive for CD163 were also positive for STAT1.ConclusionsCD163+ TAMs in oral premalignant lesions coexpress CD163 and STAT1, suggesting that the TAMs in oral premalignant lesions possess an M1 phenotype in a Th1-dominated micromilieu.

Impaired c-Fos and Polo-Like Kinase 2 Induction in the Limbic System of Fear-conditioned α-Synuclein Transgenic Mice

α-Synuclein (αSYN) is genetically and neuropathologically linked to a spectrum of neurodegenerative diseases including Parkinson’s disease, dementia with Lewy bodies, and related disorders. Cognitive impairment is recapitulated in several αSYN transgenic mouse lines. However, the mechanisms of dysfunction in affected neurons are largely unknown. Here we measured neuronal activity induced gene products in the limbic system of αSYN transgenic mice upon fear conditioning (FC). Induction of the synaptic plasticity marker c-Fos was significantly reduced in the amygdala and hippocampus of (Thy1)-h[A30P]αSYN transgenic mice in an age-dependent manner. Similarly, the neuronal activity inducible polo-like kinase 2 (Plk2) that can phosphorylate αSYN at the pathological site serine-129 was up-regulated in both brain regions upon FC. Plk2 inductions were also significantly impaired in aged (Thy1)-h[A30P]αSYN transgenic mice, both in the amygdala and hippocampus. Plk2 inductions in the amygdala after FC were paralleled by a small but significant increase in the number of neuronal cell bodies immunopositive for serine-129 phosphorylated αSYN in young but not aged (Thy1)-h[A30P]αSYN transgenic mice. In addition, we observed in the aged hippocampus a distinct type of apparently unmodified transgenic αSYN profiles resembling synaptic accumulations of αSYN. Thus, the cognitive decline observed in aged αSYN transgenic mice might be due to impairment of neurotransmission and synaptic plasticity in the limbic system by distinct αSYN species.

Cyr61 Mediates Hepatocyte Growth Factor–Dependent Tumor Cell Growth, Migration, and Akt Activation

Certain tumor cell responses to the growth factor-inducible early response gene product CCN1/Cyr61 overlap with those induced by the hepatocyte growth factor (HGF)/c-Met signaling pathway. In this study, we investigate if Cyr61 is a downstream effector of HGF/c-Met pathway activation in human glioma cells. A semiquantitative immunohistochemical analysis of 112 human glioma and normal brain specimens showed that levels of tumor-associated Cyr61 protein correlate with tumor grade (P < 0.001) and with c-Met protein expression (r(2) = 0.4791, P < 0.0001). Purified HGF rapidly upregulated Cyr61 mRNA (peak at 30 minutes) and protein expression (peak at 2 hours) in HGF(-)/c-Met(+) human glioma cell lines via a transcription- and translation-dependent mechanism. Conversely, HGF/c-Met pathway inhibitors reduced Cyr61 expression in HGF(+)/c-Met(+) human glioma cell lines in vitro and in HGF(+)/c-Met(+) glioma xenografts. Targeting Cyr61 expression with small interfering RNA (siRNA) inhibited HGF-induced cell migration (P < 0.01) and cell growth (P < 0.001) in vitro. The effect of Cyr61 on HGF-induced Akt pathway activation was also examined. Cyr61 siRNA had no effect on the early phase of HGF-induced Akt phosphorylation (Ser(473)) 30 minutes after stimulation with HGF. Cyr61 siRNA inhibited a second phase of Akt phosphorylation measured 12 hours after cell stimulation with HGF and also inhibited HGF-induced phosphorylation of the Akt target glycogen synthase kinase 3alpha. We treated preestablished subcutaneous glioma xenografts with Cyr61 siRNA or control siRNA by direct intratumoral delivery. Cyr61 siRNA inhibited Cyr61 expression and glioma xenograft growth by up to 40% in a dose-dependent manner (P < 0.05). These results identify a Cyr61-dependent pathway by which c-Met activation mediates cell growth, cell migration, and long-lasting signaling events in glioma cell lines and possibly astroglial malignancies.

Identification of DUF538 cDNA clone from Celosia cristata expressed sequences of nonstressed and stressed leaves

DUF538 domain-containing protein family consists of several plant proteins of unknown functions. This protein family has already been discovered by genome annotation tools and cloned as an inducible gene product under various environmental stress conditions. For the first time, we presented a full length DUF538 cDNA (encoding 170 amino acid residues) clone, which was randomly isolated from Celosia cristata leaf cDNA library constructed under normal growth conditions and consistently amplified from leaf cDNA populations prepared from nonstressed and drought-stressed leaves. We predicted that a DUF538 gene product can be a putative candidate for common stress-related protein (regulatory factor) in the plant system. The nucleotide and deduced amino acid sequences of the isolated clone have been submitted to EMBL data bases under accession no. AJ535713.

Juvenile Dermatomyositis

Juvenile dermatomyositis (JDM) is a rare, presumably autoimmune illness that causes proximal muscle weakness and a variety of typical cutaneous features. The study of this illness has been hampered by its rarity but, in recent years, important developments have increased our understanding of JDM. Genetic factors are likely important in the pathogenesis of JDM. These include several Human Leukocyte Antigen alleles, in particular those associated with the 8.1 ancestral haplotype and the tumor necrosis factor-alpha gene 308 polymorphism. Microchimerism, activation of plasmacytoid dendritic cells, and upregulation of type-1 interferon inducible genes also appear to play an important role in the pathogenesis of JDM. The study of JDM has also been limited by a lack of validated assessment tools. Recent work has validated the Childhood Myositis Assessment Scale and the Childhood Health Assessment Questionnaire as measures of muscle strength and function, and the Cutaneous Assessment Tool as a measure of skin disease activity and damage. Development of core sets of tools that should be used in all JDM studies has also been an important step. The use of magnetic resonance imaging and novel laboratory assessments (such as type-1 interferon inducible gene products, peripheral blood B cell and natural killer cell numbers, and myositis-associated and myositis-specific autoantibodies) are also playing an increasing role in the diagnosis and assessment of JDM. Current treatment is with corticosteroids, frequently in combination with other medications such as methotrexate or intravenous gammaglobulin. Newer therapies, such as anti-tumor necrosis factor agents and rituximab are currently being evaluated; it is not clear what role these medications will have in the future.

The ubiquitin‐proteasome system in cancer, a major player in DNA repair. Part 1: post‐translational regulation

DNA repair is a fundamental cellular function, indispensable for cell survival, especially in conditions of exposure to environmental or pharmacological effectors of DNA damage. The regulation of this function requires a flexible machinery to orchestrate the reversal of harmful DNA lesions by making use of existing proteins as well as inducible gene products. The accumulation of evidence for the involvement of ubiquitin-proteasome system (UPS) in DNA repair pathways, that is reviewed here, has expanded its role from a cellular waste disposal basket to a multi-dimensional regulatory system. This review is the first of two that attempt to illustrate the nature and interactions of all different DNA repair pathways where UPS is demonstrated to be involved, with special focus on cancer- and chemotherapy-related DNA-damage repair. In this first review, we will be presenting the proteolytic and non-proteolytic roles of UPS in the post-translational regulation of DNA repair proteins, while the second review will focus on the UPS-dependent transcriptional response of DNA repair after DNA damage and stress.

βig-h3포함 키토산 상처 도포제의 섬유아세포 증식 및 상처치유 개선 능 평가

-h3, is a TGF--induced gene product, extracellular matrix protein with 68 kDa MW(683 amino acids) and has been known for its possible roles in cell adhesion, spreading, migration and proliferation. To minimize a proteolytic degradation of -h3, -h3 incorporated chitosan sponge was prepared and its effects on fibroblast adhesion and migration were investigated. And its wound healing efficacy was evaluated in deep 2nd degree burn rabbit ear wound model. -h3 enhanced fibroblast adhesion and proliferation. In histological observation, a significant over-proliferation of epidermal regeneration was observed in -h3/chitosan dressing applied wound while epidermal regeneration was not proceeded yet in chitosan only treated wound. -h3/sponge dressing could enhance epidermal regeneration.

Activation process of macrophages afterin vitrotreatment of mouse lymphocytes with dodecylglycerol

Alkylglycerols, inflammation products of cancerous membrane lipids, efficiently activate macrophages. A brief in vitro treatment (30 min) of peritoneal cells (mixture of non-adherent and adherent cells) with a small amount (50 ng/ml) of synthetic dodecylglycerol (DDG) resulted in greatly enhanced Fc-receptor-mediated ingestion activity of macrophages. However, treatment of adherent cells (macrophages) alone with DDG produced no significant enhancement of macrophage ingestion activity, implying that macrophage activation requires a contribution of non-adherent cells. DDG-treated non-adherent cells were found to generate a macrophage-activating signal factor. Studies with a serum free-0.1% egg albumin-supplemented RPMI 1640 medium revealed that a serum factor is essential for macrophage activation process. Time course analysis of stepwise transfers of conditioned media of DDG-treated or untreated B cells and T cells revealed that DDG-treated B cells rapidly transmit a factor to untreated T cells which yield the ultimate macrophage-activating factor. This signal transmission among these cells for the macrophage activation process is too rapid to allow time for synthesis of inducible gene products. Thus, we hypothesized that a serum factor is modified by the pre-existing function of DDG-treated B cells and further modified by the pre-existing function of untreated T cells to yield macrophage-activating factor. This hypothesis was confirmed by the demonstration that DDG-treated splenic non-adherent cell ghosts modify a serum factor to yield macrophage-activating factor.

The Association between the Vascular Endothelial Growth Factor–to–Cancer Antigen 125 Ratio in Peritoneal Dialysis Effluent and the Epithelial-to-Mesenchymal Transition in Continuous Ambulatory Peritoneal Dialysis

We examined peritoneal growth factors, mesothelial mass, and epithelial-to-mesenchymal transition (EMT) in response to peritoneal exposure to peritoneal dialysate with standard and low concentrations of glucose degradation products (GDPs). We randomized 56 incident continuous ambulatory peritoneal dialysis (CAPD) patients to receive either low-GDP (30 patients) or high-GDP (standard) peritoneal dialysis (PD) solution (26 patients). The effects of the PD solutions on EMT and peritoneal growth factors in overnight dialysate effluent were compared at 1, 6, and 12 months. Assessment of EMT was performed after human peritoneal mesothelial cells (HPMCs) were cultured from overnight effluent. The low-GDP solution group showed significantly higher dialysate levels of cancer antigen 125 (CA125), fibronectin, transforming growth factor beta(TGFbeta)-induced gene product (betaig-h3), and interleukin-6 (IL-6), but the rate of EMT was significantly lower in the low-GDP solution group during the initial 12 months of CAPD treatment. After adjusting peritoneal growth factors for dialysate CA125 concentration, the low-GDP solution group showed significantly lower ratios of fibronectin/CA125, betaig-h3/CA125, IL-6/CA125, TGFbeta/CA125, and vascular endothelial growth factor (VEGF)/CA125 than did patients in the high-GDP (standard) solution group. Factors associated with higher EMT were the type of solution (high in GDPs), the mass of HPMCs (low CA125), and higher VEGF/CA125. Adjustment of dialysate VEGF for effluent CA125 revealed a significant association with EMT. It suggests that fibroblastoid transition from HPMCs could be affected by the intraperitoneal VEGF per unit mass of HPMCs.

Whole-genome expression profiling of the marine diatom Thalassiosira pseudonana identifies genes involved in silicon bioprocesses.

Formation of complex inorganic structures is widespread in nature. Diatoms create intricately patterned cell walls of inorganic silicon that are a biomimetic model for design and generation of three-dimensional silica nanostructures. To date, only relatively simple silica structures can be generated in vitro through manipulation of known diatom phosphoproteins (silaffins) and long-chain polyamines. Here, we report the use of genome-wide transcriptome analyses of the marine diatom Thalassiosira pseudonana to identify additional candidate gene products involved in the biological manipulation of silicon. Whole-genome oligonucleotide tiling arrays and tandem mass spectrometry identified transcripts for >8,000 genes, approximately 3,000 of which were not previously described and included noncoding and antisense RNAs. Gene-specific expression profiles detected a set of 75 genes induced only under low concentrations of silicon but not under low concentrations of nitrogen or iron, alkaline pH, or low temperatures. Most of these induced gene products were predicted to contain secretory signals and/or transmembrane domains but displayed no homology to known proteins. Over half of these genes were newly discovered, identified only through the use of tiling arrays. Unexpectedly, a common set of 84 genes were induced by both silicon and iron limitations, suggesting that biological manipulation of silicon may share pathways in common with iron or, alternatively, that iron may serve as a required cofactor for silicon processes. These results provide insights into the transcriptional and translational basis for the biological generation of elaborate silicon nanostructures by these ecologically important microbes.

The TGF- -induced gene product, ig-h3: its biological implications in peritoneal dialysis

TGF-beta is involved in peritoneal changes during long-term peritoneal dialysis (PD). TGF-beta induces betaig-h3 in several cell lines, and betaig-h3 may be a marker for biologically active TGF-beta. However, no study has reported induction of betaig-h3 in human peritoneal mesothelial cells (HPMCs) or its involvement in PD-related peritoneal membrane changes. We used cultured HPMCs to investigate the biological roles of betaig-h3 during mesothelial cell injury and repair, employing the adhesion, spreading, scratching and cell migration assays. Changes in betaig-h3 expression after high glucose exposure in vivo were also evaluated using an animal chronic PD model. In vitro, TGF-beta1 induced betaig-h3 in cultured HPMCs, and betaig-h3-mediated mesothelial cell adhesion occurred via alphavbeta3 integrin. betaig-h3 enhanced mesothelial cell adhesion and migration and, in part, wound healing during mesothelial cell injury. The animal study demonstrated that compared to the control group, betaig-h3 concentrations in the dialysate effluent increased in the dialysis group with alterations in peritoneal structure and function during PD, and betaig-h3 positively correlated with peritoneal solute transport. Immunohistochemical and immunoblotting results showed that betaig-h3 localizes in the mesothelium and submesothelial matrix of the parietal peritoneum, and in the vascular endothelium of omentum. betaig-h3 protein expression was higher in the dialysis group. In vitro, betaig-h3 induced by TGF-beta1 in HPMCs improved adhesion and migration of HPMCs during wound healing. In the chronic infusion model of PD, betaig-h3 played a role in the functional deterioration of the peritoneal membrane, which is associated with fibrosis.

Cardiac involvement in patients with sorafenib or sunitinib treatment for metastatic renal cell carcinoma

5110 Background: Tyrosine-kinase inhibitors (TKI) of the VEGF and PDGF-receptor have significant clinical activity in patients with renal cell carcinoma (RCC). These agents target the VHL-hypoxia-inducible gene pathway and lead to inhibition of hypoxia- inducible factor (HIF)-induced gene products. Physiologically, HIF-1 related gene products are important mediators of myocardial response to ischemia, myocardial remodeling, peri-infarct vascularisation and vascular permeability. The aim of this prospective observational study was to investigate clinical and biochemical signs of myocardial damage in patients undergoing TKI-treatment for RCC. Methods: 73 consecutive patients (median age 65, range 44–68) intended for TKI treatment were analyzed for medical history of coronary artery disease (CAD) and risk-factors. Measurements of biochemical markers of cardiac damage (creatine kinase MB -CK-MB- and cardiac troponin T -cTNT-) and electrocardiogram (ECG) were performed before treatment. In patients developing cardiac symptoms during TKI treatment and/or at occurrence of CK-MB or TNT elevations, changes in ECG were analyzed and patients underwent echocardiography. Results: All patients had normal CK-MB and TNT levels at baseline. 17 patients (23%) developed (week 2–32 of treatment) significant CK-MB elevation, (TNT n=5), with clinical symptoms in 7 patients. No patient had uncontrolled hypertension. Detailed ECG’s comparison before and during treatment revealed significant changes in 10 out of 17 patients, such as ST-segment depression or elevation, T-wave changes and symptomatic AV-conduction disturbance, requiring pacemaker-implantation. 3 patients underwent coronary angiography with one patient showing acute coronary artery occlusion and myocardial infarction. 6 out of 17 patients had abnormal findings on echocardiography, such as reduced left ventricular function Conclusions: TKI-induced HIF-inhibition may be associated with severe myocardial damage. The underlying mechanism may not necessarily be caused by overt coronary artery occlusion. ECG-changes and biochemical markers are the most important indicators in the preclinical stage. Therefore, careful cardiac monitoring during TKI-treatment is strongly recommended. No significant financial relationships to disclose.

Interphase-specific Phosphorylation-mediated Regulation of Tubulin Dimer Partitioning in Human Cells

The microtubule cytoskeleton is differentially regulated by a diverse array of proteins during interphase and mitosis. Op18/stathmin (Op18) and microtubule-associated protein (MAP)4 have been ascribed opposite general microtubule-directed activities, namely, microtubule destabilization and stabilization, respectively, both of which can be inhibited by phosphorylation. Here, using three human cell models, we depleted cells of Op18 and/or MAP4 by expression of interfering hairpin RNAs and we analyzed the resulting phenotypes. We found that the endogenous levels of Op18 and MAP4 have opposite and counteractive activities that largely govern the partitioning of tubulin dimers in the microtubule array at interphase. Op18 and MAP4 were also found to be the downstream targets of Ca(2+)- and calmodulin-dependent protein kinase IV and PAR-1/MARK2 kinase, respectively, that control the demonstrated counteractive phosphorylation-mediated regulation of tubulin dimer partitioning. Furthermore, to address mechanisms regulating microtubule polymerization in response to cell signals, we developed a system for inducible gene product replacement. This approach revealed that site-specific phosphorylation of Op18 is both necessary and sufficient for polymerization of microtubules in response to the multifaceted signaling event of stimulation of the T cell antigen receptor complex, which activates several signal transduction pathways.