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Colorectal cancer (CRC) is one of the most common cancers in the world. Here, we undertook an analysis of microarray datasets consisting of colon biopsies of healthy subjects and of patients affected by CRC, in order to analyze the expression levels of Chitinase domain-containing protein 1 (CHID1) and to correlate them with the clinical data available in the datasets. Analysis of expression levels showed a significant increase of CHID1 in CRC biopsies compared to the mucosa of healthy subjects. Patients' stratification by TNM staging revealed significant increases in CHID1 expression levels as the disease progressed. Furthermore, we found that mutated BRAF patients exhibit higher levels of CHID1 expression. Patients with a poor surviving prognosis at 5 years expressed high levels of CHID1 compared to wild-type. The histochemical analysis carried out by the Human Protein Atlas web tool documented moderate to strong-intensity staining detection of CHID1 protein in CRC biopsies. Furthermore, CRC patients were selected and clustered into two groups, high and low CHID1 expression levels (HCEL and LCEL). We obtained two signatures, the genes significant positive (GSPC-CHID1) and negative (GSNC-CHID1) correlated to CHID1 expression levels. The genomic deconvolution analysis between the GSPC-CHID1, GSNC-CHID1, and 17 cell immunological signatures, highlighted the potential infiltration of Macrophages M0 in HCEL patients, and potential infiltration of Macrophages M1 cells in LCEL patients. In addition, the signature GSPC-CHID1 expressed unfavorable genes to the CRC patient's survival. Mirror results were obtained for the GSNC-CHID1 signature. From the outcome of our investigation, it is possible to conclude that HCEL are associated with an unfavorable prognosis for CRC patients. more...

Alpha-enolase is a key glycolytic enzyme that plays a functional role in several physiological processes depending on the cellular localization. The enzyme is mainly localized in the cytoplasm whereas an alternative translated form, named MBP-1, is predominantly nuclear. The MBP-1 protein has been characterized as a c-Myc promoter binding protein that negatively controls transcription. In the present study, we identified the kelch protein NS1-BP as one of the alpha-enolase/MBP-1 partners by using a yeast two-hybrid screening. Although NS1-BP has been originally described as a protein mainly localized in the nucleus, we provide evidence that NS1-BP also interacts with actin in human cells, as reported for most kelch-containing proteins. Here we showed that alpha-enolase and MBP-1 associate with NS1-BP in vitro and in vivo by GST pull-down assays and coimmunoprecipitation experiments; subsequent immunofluorescent staining confirmed colocalization of the proteins within the cells. Furthermore, functional analyses performed by cotransfection assays revealed that NS1-BP enhances the inhibitory effect exerted by MBP-1 on c- Myc promoter. In mammalian cells, the overexpression of both proteins resulted in an increased repression of basal c-Myc transcription and consistently affected the steady state levels of endogenous c-Myc mRNA. These findings further support the distinct roles of alpha-enolase and its MBP-1 variant in maintaining cell homeostasis. Moreover, our data suggest a novel function for NS1-BP in the control of cell proliferation. © 2007 Elsevier B.V. All rights reserved. more...

Azimuthally controlled grating-coupling surface plasmon resonance (φ ≠ 0° GC-SPR) is known to exhibit refractive index sensitivity (SRI) up to one order of magnitude higher with respect to classic φ = 0° GC-SPR. Here, grated gold surfaces (period of 500 nm and amplitude of 40 nm) were covered with different amounts of thiol-poly(ethylene oxide) (PEO, Mw 5 kDa) through a 24 h functionalization process using polymer solutions at increasing concentration (from 500 nM to 2.5 mM). The GC-SPR output signal generated by each PEO layer was measured in both null and rotated configuration. The number of PEO molecules adsorbed onto each surface (cm−2) and the corresponding molecular surface densities (σ [ng/cm2]) were calculated from the angle shifts collected after PEO film formation, by combining the model shown by Jung and the theory of PEO layer formation. In the end, we obtained a calibration curve that correlates the amount of PEO tethered onto the surface with the SPR measurement output signal, that will be useful for future reference. At the PEO surface saturation condition (PEO σ = 592 ng/cm2, obtained using PEO concentration ≥2 mM), a SRI of 603°/RIU was reached (φ = 45°), which is ≈10-fold higher than what obtained through the classic GC-SPR (φ = 0°). This enhanced instrumental sensitivity allowed detecting tiny amounts of molecules adsorbed onto the surface (3.1 ng vs 59.7 ng of classic φ = 0° GC-SPR), a property that is extremely valuable for the application of SPR sensing to quantitative determinations. more...

The common sea star Asterias rubens represents a key-species of the North-Eastern Atlantic macro benthic community. The cells of their immune system, known as coelomocytes, are the first line of defence against environmental hazards. Here, we report the results of investigations on the immune cells response of sea stars exposed to marine environmental pollution for long periods. We show that levels of the heat shock cognate protein 70 (HSC70) in coelomocytes from A. rubens, which were collected during a field study in the S?rfjord (North Sea, SW coast of Norway) along a contamination gradient, are directly associated with the long-term accumulation of Cd, Cu heavy metals exclusively in the tegument. Conversely, Pb and Zn accumulation in the tegument did not relate to HSC70 levels and none of the metals were found accumulated in the pyloric coeca. In addition the coelomocytes from A. rubens, collected in high and low metal impacted stations were examined by a proteomic approach using two-dimensional electrophoresis (2DE). By comparison of the proteomic maps, we observed that 31 protein spots differed in their relative abundance, indicating a gene expression response to the metal mixture exposure. All together, our results confirm that the echinoderm immune cells are a suitable model for the assessment of long-term exposure to environmental pollution, moreover that the increased level of HSC70 can be considered a signal of an acquired tolerance within a large spectrum of protein profile changes occurring in response to metal contamination. more...

A transcription suppressor element (sequence -481 to -320) containing a G-rich motif (designated GTG) and a newly identified CAT-rich motif (designated CATR) was previously shown to modulate expression of the mouse cytochrome c oxidase Vb gene during myogenesis. Here, we show that the GTG element is critical for transcription activation in both undifferentiated and differentiated myocytes. Mutations of the CATR motif abolished transcription repression in myoblasts while limiting transcription activation in differentiated myotubes, suggesting contrasting functional attributes of this DNA motif at different stages of myogenesis. Results show that the activity of the transcription suppressor motif is modulated by an orchestrated interplay between ubiquitous transcription factors: ZBP-89, YY-1, and a member of the heterogeneous nuclear ribonucleoprotein D-like protein (also known as JKTBP1) family. In undifferentiated muscle cells, GTG motif-bound ZBP-89 physically and functionally interacted with CATR motif-bound YY-1 to mediate transcription repression. In differentiated myotubes, heterogeneous nuclear ribonucleoprotein D-like protein/JKTBP1 bound to the CATR motif exclusive of YY-1 and interacted with ZBP-89 in attenuating repressor activity, leading to transcription activation. Our results show a novel mechanism of protein factor switching in transcription regulation of the cytochrome c oxidase Vb gene during myogenesis. more...

We have characterized the genomic structure of the mouse Zfp148 gene encoding Beta-Enolase Repressor Factor-1 (BERF-1), a Kruppel-like zinc finger protein involved in the transcriptional regulation of several genes, which is also termed ZBP-89, BFCOL1. The cloned Zfp148 gene spans 110 kb of genomic DNA encompassing the 5′-end region, 9 exons, 8 introns, and the 3′-untranslated region. The promoter region displays the typical features of a housekeeping gene: a high G+C content and the absence of canonical TATA and CAAT boxes consistent with the multiple transcription initiation sites determined by primary extension analysis. Computer-assisted search in the human genome database allowed us to determine that the same genomic structure with identical intron–exon organization is conserved in the human homologue ZNF 148. Functional analysis of the 5′-flanking sequence of the mouse gene indicated that the region from nucleotide −205 to +144, relative to the major transcription start site, contains cis-regulatory elements that promote basal expression. Such sequences and the overall promoter architecture are highly conserved in the human gene. Furthermore, we show that the complex transcription pattern of the Zfp148 gene might be due to a combination of alternative splicing and differential polyadenylation sites utilization. more...

We have previously identified a muscle-specific enhancer within the first intron of the human beta enolase gene. Present in this enhancer are an A/T-rich box that binds MEF-2 protein(s) and a G-rich box (AGTGGGGGAGGGGGCTGCG) that interacts with ubiquitously expressed factors. Both elements are required for tissue-specific expression of the gene in skeletal muscle cells. Here, we report the identification and characterization of a Kruppel-like zinc finger protein, termed beta enolase repressor factor 1, that binds in a sequence-specific manner to the G-rich box and functions as a repressor of the beta enolase gene transcription in transient transfection assays. Using fusion polypeptides of beta enolase repressor factor 1 and the yeast GAL4 DNA-binding domain, we have identified an amino-terminal region responsible for the transcriptional repression activity, whereas a carboxyl-terminal region was shown to contain a potential transcriptional activation domain. The expression of this protein decreases in developing skeletal muscles, correlating with lack of binding activity in nuclear extract from adult skeletal tissue, in which novel binding activities have been detected. These results suggest that in addition to the identified factor, which functionally acts as a negative regulator and is enriched in embryonic muscle, the G-rich box binds other factors, presumably exerting a positive control on transcription. The interplay between factors that repress or activate transcription may constitute a developmentally regulated mechanism that modulates beta enolase gene expression in skeletal muscle. more...

Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix transcription factor which is expressed when mammalian cells are subjected to hypoxia and which activates transcription of genes encoding erythropoietin, vascular endothelial growth factor, and other proteins that are important for maintaining oxygen homeostasis. Previous studies have provided indirect evidence that HIF-1 also regulates transcription of genes encoding glycolytic enzymes. In this paper we characterize hypoxia response elements in the promoters of the ALDA, ENO1, and Ldha genes. We demonstrate that HIF-1 plays an essential role in activating transcription via these elements and show that although absolutely necessary, the presence of a HIF-1 binding site alone is not sufficient to mediate transcriptional responses to hypoxia. Analysis of hypoxia response elements in the ENO1 and Ldha gene promoters revealed that each contains two functionally-essential HIF-1 sites arranged as direct and inverted repeats, respectively. Our data establish that functional hypoxia-response elements consist of a pair of contiguous transcription factor binding sites at least one of which contains the core sequence 5'-RCGTG-3' and is recognized by HIF-1. These results provide further evidence that the coordinate transcriptional activation of genes encoding glycolytic enzymes which occurs in hypoxic cells is mediated by HIF-1. more...

At least three genes encode the different isoforms of the glycolytic enzyme enolase. We have isolated the gene for the human gamma- or neuron-specific enolase and determined the nucleotide sequence from upstream to the 5' end to beyond the polyadenylation site. The gene contains 12 exons distributed over 9213 nucleotides. Introns occur at positions identical to those reported for the homologous rat gene, as well as for the human alpha- or nonneuronal enolase gene, supporting the existence of a single ancestor for the members of this gene family. Primer extension analysis indicates that the gene has multiple start sites. The putative promoter region lacks canonical TATA and CAAT boxes, is very G + C-rich, and contains several potential regulatory sequences. Furthermore, an inverted Alu sequence is present approximately 572 nucleotides upstream of the major start site. A comparison of the 5'-flanking region of the human gamma-enolase gene with the same region of the rat gene revealed a high degree of sequence conservation. more...

In patients with MM we measured selected parameters of MBD in comparison with the stage of the disease according to Durie-Salmon (DS) and according to International Staging System (ISS). In MGUS we assessed the relationship of the parameters to the risk of transformation to MM based on the Mayo clinic stratification system. Weassessed following parameters ofMBD:hepatocyte growth factor (HGF, Human HGF Quantine ELISA), macrophage inflammatory factor alpha (MIP-1 , Human CCL3/MIP-1 Quantikine ELISA), syndecan-1 (Human Syndecan-1 (CD138) ELISA), osteoprotegerin (OPG,HumanOsteoprotegerin ELISA), Activin A (Human Activin A Quantikine ELISA), Dickkopf-related protein-1 (DKK1, Human Dkk-1 Quantikine ELISA), Annexin A2 (Human Annexin A2 ELISA kit), nuclear factor kappa B (NFB, Human Nuclear factor-kappa B). For statistical estimation we used Kruskal-Wallis test and MannWhitney U post hoc test with Bonferroni correction at p < 0,05. Results: Within the comparison of MM and MGUS we found significant differences in serum levels of HGF (median 1⁄4 M 2997 vs 1757pg/mL), MIP-1 (M 25,5 vs 21,1pg/mL), syndecan-1 (M 77,3 vs 24,2ng/mL), and NFB (M 0,61 vs 0,26ng/mL). There was a trend towards increasing serum levels of these parameters when comparing MGUS, SMM and AMM, the difference between MGUS and SMM and the difference between SMM and AMM were not, however, statistically significant. With regard to the activity of the disease there was a significant relationship of syndecan-1 to DS, and significant relationship of HGF, MIP-1, syndecan-1, OPG and Activin A to ISS. Neither of the parameters had significant relationship to Mayo clinic risk stratification in MGUS individuals. Conclusions: The selected parameters of MBD showed relationship to the activity of MM with certain discriminatory potential of MM and MGUS. The most perspective parameters were HGF, MIP-1, syndecan-1, OPG, Activin A and NFB. There were no differences in these parameters in MGUS with regard to the risk of transformation into MM. Supported by the grant NT 14393. more...

The human gene encoding the muscle-specific β-enolase has been isolated. The β-enolase gene was mapped to chromosome 17 by analysis of a panel of rodent-human somatic cell hybrids. The gene was further localized to the short arm and tentatively to the region 17pter-p11 by analysis of cell hybrids and transfectant cell lines carrying different portions of chromosome 17. more...