The role of the BMP signaling antagonist noggin in the development of prostate cancer osteolytic bone metastasis

Members of the BMP and Wnt protein families play a relevant role in physiologic and pathologic bone turnover. Extracellular antagonists are crucial for the modulation of their activity. Lack of expression of the BMP antagonist noggin by osteoinductive, carcinoma-derived cell lines is a determinant of the osteoblast response induced by their bone metastases. In contrast, osteolytic, carcinoma-derived cell lines express noggin constitutively. We hypothesized that cancer cell-derived noggin may contribute to the pathogenesis of osteolytic bone metastasis of solid cancers by repressing bone formation. Intra-osseous xenografts of PC-3 prostate cancer cells induced osteolytic lesions characterized not only by enhanced osteoclast-mediated bone resorption, but also by decreased osteoblast-mediated bone formation. Therefore, in this model, uncoupling of the bone remodeling process contributes to osteolysis. Bone formation was preserved in the osteolytic lesions induced by noggin-silenced PC-3 cells, suggesting that cancer cell-derived noggin interferes with physiologic bone coupling. Furthermore, intra-osseous tumor growth of noggin-silenced PC-3 cells was limited, most probably as a result of the persisting osteoblast activity. This investigation provides new evidence for a model of osteolytic bone metastasis where constitutive secretion of noggin by cancer cells mediates inhibition of bone formation, thereby preventing repair of osteolytic lesions generated by an excess of osteoclast-mediated bone resorption. Therefore, noggin suppression may be a novel strategy for the treatment of osteolytic bone metastases.

GLP-2 receptors in human disease: high expression in gastrointestinal stromal tumors and Crohn's disease

Peptide hormones of the glucagon-like peptide (GLP) family play an increasing clinical role, as reported for GLP-1 in diabetes therapy and insulinoma diagnostics. GLP-2, despite its known trophic and anti-inflammatory intestinal actions translated into preliminary clinical studies using the GLP-2 analogue teduglutide for treatment of short bowel syndrome and Crohn's disease, remains poorly characterized in terms of expression of its receptor in tissues of interest. Therefore, the GLP-2 receptor expression was assessed in 237 tumor and 148 non-neoplastic tissue samples with in vitro receptor autoradiography. A GLP-2 receptor expression was present in 68% of gastrointestinal stromal tumors (GIST). Furthermore, GLP-2 receptors were identified in the intestinal myenteric plexus, with significant up-regulation in active Crohn's disease. The GLP-2 receptors in GIST may be used for clinical applications like in vivo targeting with radiolabelled GLP-2 analogues for imaging and therapy. Moreover, the over-expressed GLP-2 receptor in the myenteric plexus may represent the morphological correlate of the clinical target of teduglutide in Crohn's disease.

Interleukin-6 is an efficacious marker of axonal transport disruption during experimental glaucoma and stimulates neuritogenesis in cultured retinal ganglion cells

It is increasingly recognised that chronically activated glia contribute to the pathology of various neurodegenerative diseases, including glaucoma. One means by which this can occur is through the release of neurotoxic, proinflammatory factors. In the current study, we therefore investigated the spatio-temporal patterns of expression of three such cytokines, IL-1β, TNFα and IL-6, in a validated rat model of experimental glaucoma. First, only weak evidence was found for increased expression of IL-1β and TNFα following induction of ocular hypertension. Second, and much more striking, was that robust evidence was uncovered showing IL-6 to be synthesised by injured retinal ganglion cells following elevation of intraocular pressure and transported in an orthograde fashion along the nerve, accumulating at sites of axonal disruption in the optic nerve head. Verification that IL-6 represents a novel marker of disrupted axonal transport in this model was obtained by performing double labelling immunofluorescence with recognised markers of fast axonal transport. The stimulus for IL-6 synthesis and axonal transport during experimental glaucoma arose from axonal injury rather than ocular hypertension, as the response was identical after optic nerve crush and bilateral occlusion of the carotid arteries, each of which is independent of elevated intraocular pressure. Moreover, the response of IL-6 was not a generalised feature of the gp130 family of cytokines, as it was not mimicked by another family member, ciliary neurotrophic factor. Finally, further study suggested that IL-6 may be an early part of the endogenous regenerative response as the cytokine colocalised with growth-associated membrane phosphoprotein-43 in some putative regenerating axons, and potently stimulated neuritogenesis in retinal ganglion cells in culture, an effect that was additive to that of ciliary neurotrophic factor. These data comprise clear evidence that IL-6 is actively involved in the attempt of injured retinal ganglion cells to regenerate their axons.

Hexose, sialic acid and sialyllactose concentrations in the milk of dairy and non-dairy breed cows

It has been suggested that the ratio of lactose to milk oligosaccharides in mammalian milk/colostrum is based on the ratio of expression of a-lactalbumin and glycosyltransferases in the mammary epithelial cells. It has also been suggested that the high secretion of milk in dairy breed cows has been acquired by a high expression of a-lactalbumin expression. As there is a large difference of milk secretion level between dairy and non dairy breed cows, there may be a difference in the ratio of lactose to milk oligosaccharides in milks between dairy and non dairy breed cows. In this study, the concentrations of hexose, sialic acid as well as sialyllactoses, which are representative bovine milk oligosaccharides, were determined in the milks of dairy and non dairy breed cows. The concentration of hexose was significantly higher in the milks of non dairy breed cows than that of dairy breed cows, but there were no significant differences with respect to sialic acid and sialyllactose. The significant difference of the ratio of the concentrations of 3'- and 6'-sialyllactose to total hexose in milk was not observed between dairy and non dairy cows.

eIF4E is an important determinant of adhesion and pseudohyphal growth of the yeast S. cerevisiae

eIF4E, the cytoplasmatic cap-binding protein, is required for efficient cap-dependent translation. We have studied the influence of mutations that alter the activity and/or expression level of eIF4E on haploid and diploid cells in the yeast S. cerevisiae. Temperature-sensitive eIF4E mutants with reduced levels of expression and reduced cap-binding affinity clearly show a loss in haploid adhesion and diploid pseudohyphenation upon starvation for nitrogen. Some of these mutations affect the interaction of the cap-structure of mRNAs with the cap-binding groove of eIF4E. The observed reduction in adhesive and pseudohyphenating properties is less evident for an eIF4E mutant that shows reduced interaction with p20 (an eIF4E-binding protein) or for a p20-knockout mutant. Loss of adhesive and pseudohyphenating properties was not only observed for eIF4E mutants but also for knockout mutants of components of eIF4F such as eIF4B and eIF4G1. We conclude from these experiments that mutations that affect components of the eIF4F-complex loose properties such as adhesion and pseudohyphal differentiation, most likely due to less effective translation of required mRNAs for such processes.

Eugene Onegin As A Bridge Between Worlds: Aleksandr Pushkin As Translator Of The 19Th-Century Russian Experience

In my thesis, I use literary criticism, knowledge of Russian, and elements of translation theory to study the seminal poet of the Russian literary tradition ¿ Aleksandr Pushkin. In his most famous work, Eugene Onegin, Pushkin explores the cultural and linguistic divide in place at the turn of the 19th century in Russia. Pushkin stands on the peripheries of several colliding worlds; never fully committing to any of them, he acts as a translator between various realms of the 19th-century Russian experience. Through his narrator, he adeptly occupies the voices, styles, and modes of expression of various characters, displaying competency in all realms of Russian life. In examining Tatiana, his heroine, the reader witnesses her development as analogous to the author¿s. At the center of the text stands the act of translation itself: as the narrator ¿translates¿ Tatiana¿s love letter from French to Russian, the author-narrator declares his function as a mediator, not only between languages, but also between cultures, literary canons, social classes, and identities. Tatiana, as both main character and the narrator¿s muse, emerges as the most complex figure in the novel, and her language manifests itself as the most direct and capable of sincerity in the novel. The elements of Russian folklore that are incorporated into her language speak to Pushkin¿s appreciation for the rich Russian folklore tradition. In his exaltation of language considered to be ¿common¿, ¿low¿ speech is juxtaposed with its lofty counterpart; along the way, he incorporates myriad foreign borrowings. An active creator of Russia¿s new literary language, Pushkin traverses linguistic boundaries to synthesize a fragmented Russia. In the process, he creates a work so thoroughly tied to language and entrenched in complex cultural traditions that many scholars have argued for its untranslatability.

A GABA(A) receptor of defined subunit composition and positioning: concatenation of five subunits

We show that the five subunits of a gamma-aminobutyric acid type A receptor (GABA(A) receptor) can be concatenated to yield a functional receptor. This concatenated receptor alpha(1)-beta(2)-alpha(1)-gamma(2)-beta(2) has the advantage of a known subunit arrangement. Most of its functional properties are not significantly different from a receptor formed by individual subunits. Extent of expression amounted to about 40% of that of non-concatenated receptors in Xenopus oocytes, after injection of oocytes with comparable amounts of cRNA coding for concatenated and non-concatenated receptors. The ability to express receptors consisting of five subunits enables detailed studies of GABA(A) receptor subtype selective compounds.

Gene expression in cortex and hippocampus during acute pneumococcal meningitis

BACKGROUND: Pneumococcal meningitis is associated with high mortality (approximately 30%) and morbidity. Up to 50% of survivors are affected by neurological sequelae due to a wide spectrum of brain injury mainly affecting the cortex and hippocampus. Despite this significant disease burden, the genetic program that regulates the host response leading to brain damage as a consequence of bacterial meningitis is largely unknown.We used an infant rat model of pneumococcal meningitis to assess gene expression profiles in cortex and hippocampus at 22 and 44 hours after infection and in controls at 22 h after mock-infection with saline. To analyze the biological significance of the data generated by Affymetrix DNA microarrays, a bioinformatics pipeline was used combining (i) a literature-profiling algorithm to cluster genes based on the vocabulary of abstracts indexed in MEDLINE (NCBI) and (ii) the self-organizing map (SOM), a clustering technique based on covariance in gene expression kinetics. RESULTS: Among 598 genes differentially regulated (change factor > or = 1.5; p < or = 0.05), 77% were automatically assigned to one of 11 functional groups with 94% accuracy. SOM disclosed six patterns of expression kinetics. Genes associated with growth control/neuroplasticity, signal transduction, cell death/survival, cytoskeleton, and immunity were generally upregulated. In contrast, genes related to neurotransmission and lipid metabolism were transiently downregulated on the whole. The majority of the genes associated with ionic homeostasis, neurotransmission, signal transduction and lipid metabolism were differentially regulated specifically in the hippocampus. Of the cell death/survival genes found to be continuously upregulated only in hippocampus, the majority are pro-apoptotic, while those continuously upregulated only in cortex are anti-apoptotic. CONCLUSION: Temporal and spatial analysis of gene expression in experimental pneumococcal meningitis identified potential targets for therapy.

The alpha and beta subunits of the metalloprotease meprin are expressed in separate layers of human epidermis, revealing different functions in keratinocyte proliferation and differentiation

The zinc endopeptidase meprin (EC 3.4.24.18) is expressed in brush border membranes of intestine and kidney tubules, intestinal leukocytes, and certain cancer cells, suggesting a role in epithelial differentiation and cell migration. Here we show by RT-PCR and immunoblotting that meprin is also expressed in human skin. As visualized by immunohistochemistry, the two meprin subunits are localized in separate cell layers of the human epidermis. Meprin alpha is expressed in the stratum basale, whereas meprin beta is found in cells of the stratum granulosum just beneath the stratum corneum. In hyperproliferative epidermis such as in psoriasis vulgaris, meprin alpha showed a marked shift of expression from the basal to the uppermost layers of the epidermis. The expression patterns suggest distinct functions for the two subunits in skin. This assumption is supported by diverse effects of recombinant meprin alpha and beta on human adult low-calcium high-temperature keratinocytes. Here, beta induced a dramatic change in cell morphology and reduced the cell number, indicating a function in terminal differentiation, whereas meprin alpha did not affect cell viability, and may play a role in basal keratinocyte proliferation.

GPR87 is an overexpressed G-protein coupled receptor in squamous cell carcinoma of the lung

Lung cancer is the leading cause of cancer death worldwide. The overall 5-year survival after therapy is about 16% and there is a clear need for better treatment options, such as therapies targeting specific molecular structures. G-protein coupled receptors (GPCRs), as the largest family of cell surface receptors, represent an important group of potential targets for diagnostics and therapy. We therefore used laser capture microdissection and GPCR-focused Affymetrix microarrays to examine the expression of 929 GPCR transcripts in tissue samples of 10 patients with squamous cell carcinoma and 7 with adenocarcinoma in order to identify novel targets in non-small cell lung carcinoma (NSCLC). The relative gene expression levels were calculated in tumour samples compared to samples of the neighbouring alveolar tissue in every patient. Based on this unique study design, we identified 5 significantly overexpressed GPCRs in squamous cell carcinoma, in the following decreasing order of expression: GPR87 > CMKOR1 > FZD10 > LGR4 > P2RY11. All are non-olfactory and GRAFS (glutamate, rhodopsin, adhesion, frizzled/taste2, secretin family) classified. GPR87, LGR4 and CMKOR1 are orphan receptors. GPR87 stands out as a candidate for further target validation due to its marked overexpression and correlation on a mutation-based level to squamous cell carcinoma.

DISSECTION OF STRESS RESPONSE NETWORKS REGULATING MULTIPLE STRESSES IN RICE

Important food crops like rice are constantly exposed to various stresses that can have devastating effect on their survival and productivity. Being sessile, these highly evolved organisms have developed elaborate molecular machineries to sense a mixture of stress signals and elicit a precise response to minimize the damage. However, recent discoveries revealed that the interplay of these stress regulatory and signaling molecules is highly complex and remains largely unknown. In this work, we conducted large scale analysis of differential gene expression using advanced computational methods to dissect regulation of stress response which is at the heart of all molecular changes leading to the observed phenotypic susceptibility. One of the most important stress conditions in terms of loss of productivity is drought. We performed genomic and proteomic analysis of epigenetic and miRNA mechanisms in regulation of drought responsive genes in rice and found subsets of genes with striking properties. Overexpressed genesets included higher number of epigenetic marks, miRNA targets and transcription factors which regulate drought tolerance. On the other hand, underexpressed genesets were poor in above features but were rich in number of metabolic genes with multiple co-expression partners contributing majorly towards drought resistance. Identification and characterization of the patterns exhibited by differentially expressed genes hold key to uncover the synergistic and antagonistic components of the cross talk between stress response mechanisms. We performed meta-analysis on drought and bacterial stresses in rice and Arabidopsis, and identified hundreds of shared genes. We found high level of conservation of gene expression between these stresses. Weighted co-expression network analysis detected two tight clusters of genes made up of master transcription factors and signaling genes showing strikingly opposite expression status. To comprehensively identify the shared stress responsive genes between multiple abiotic and biotic stresses in rice, we performed meta-analyses of microarray studies from seven different abiotic and six biotic stresses separately and found more than thirteen hundred shared stress responsive genes. Various machine learning techniques utilizing these genes classified the stresses into two major classes' namely abiotic and biotic stresses and multiple classes of individual stresses with high accuracy and identified the top genes showing distinct patterns of expression. Functional enrichment and co-expression network analysis revealed the different roles of plant hormones, transcription factors in conserved and non-conserved genesets in regulation of stress response.

OCT4 expression in human non-small cell lung cancer: implications for therapeutic intervention

Here we investigate the expression of OCT4 human lung adenocarcinoma and bronchioloalveolar carcinoma (BAC) tumor biopsies and tumor-derived primary cell cultures. OCT4 has been detected in several human tumors suggesting a potentially critical role in tumorigenesis. We assessed the presence of OCT4 in clinical tumor samples of both adenocarcinoma and BAC at the cellular and transcriptional levels, respectively. Furthermore, we evaluated tumor-derived cell cultures for potential differences in OCT4 expression. Immunohistochemical analysis depicted OCT4 in 2 of 8 adenocarcinoma tumor samples and 3 of 5 BAC tumor samples, with no apparent difference in the degree of expression among the sections examined. These results were validated by transcript analysis. Flow cytometric assessment of 11 adenocarcinoma-derived cell cultures and 3 BAC-derived cell cultures revealed significantly higher OCT4 expression in adenocarcinoma tumors compared to their normal counterparts. This, however, was not observed in the BAC cultures. Comparative studies of OCT4 in adenocarcinoma and BAC tumor cell cultures demonstrated a dramatically higher expression in the former. The expression of OCT4 may represent a specific and effective target for therapeutic intervention in adenocarcinoma and BAC. In addition, the aberrant expression and distribution of OCT4 may indicate important parameters concerning the differences between adenocarcinoma and BAC.

Use of concatamers to study GABAA receptor architecture and function: application to delta-subunit-containing receptors and possible pitfalls

Many membrane proteins, including the GABA(A) [GABA (gamma-aminobutyric acid) type A] receptors, are oligomers often built from different subunits. As an example, the major adult isoform of the GABA(A) receptor is a pentamer built from three different subunits. Theoretically, co-expression of three subunits may result in many different receptor pentamers. Subunit concatenation allows us to pre-define the relative arrangement of the subunits. This method may thus be used to study receptor architecture, but also the nature of binding sites. Indeed, it made possible the discovery of a novel benzodiazepine site. We use here subunit concatenation to study delta-subunit-containing GABA(A) receptors. We provide evidence for the formation of different functional subunit arrangements in recombinant alpha(1)beta(3)delta and alpha(6)beta(3)delta receptors. As with all valuable techniques, subunit concatenation has also some pitfalls. Most of these can be avoided by carefully titrating and minimizing the length of the linker sequences joining the two linked subunits and avoiding inclusion of the signal sequence of all but the N-terminal subunit of a multi-subunit construct. Maybe the most common error found in the literature is that low expression can be overcome by simply overloading the expression system with genetic information. As some concatenated constructs result by themselves in a low level of expression, this erroneous assembly leading to receptor function may be promoted by overloading the expression system and leads to wrong conclusions.

Virtues and Perils of Anonymity: Should Intermediaries Bear the Burden?

On October 10, 2013, the Chamber of the European Court of Human Rights (ECtHR) handed down a judgment (Delfi v. Estonia) condoning Estonia for a law which, as interpreted, held a news portal liable for the defamatory comments of its users. Amongst the considerations that led the Court to find no violation of freedom of expression in this particular case were, above all, the inadequacy of the automatic screening system adopted by the website and the users’ option to post their comments anonymously (i.e. without need for prior registration via email), which in the Court’s view rendered the protection conferred to the injured party via direct legal action against the authors of the comments ineffective. Drawing on the implications of this (not yet final) ruling, this paper discusses a few questions that the tension between the risk of wrongful use of information and the right to anonymity generates for the development of Internet communication, and examines the role that intermediary liability legislation can play to manage this tension.