Goat uromodulin promoter directs kidney-specific expression of GFP gene in transgenic mice

BACKGROUND: Uromodulin is the most abundant protein found in the urine of mammals. In an effort to utilize the uromodulin promoter in order to target recombinant proteins in the urine of transgenic animals we have cloned a goat uromodulin gene promoter fragment (GUM promoter) and used it to drive expression of GFP in the kidney of transgenic mice. RESULTS: The GUM-GFP cassette was constructed and transgenic mice were generated in order to study the promoter's tissue specificity, the GFP kidney specific expression and its subcellular distribution. Tissues collected from three GUM-GFP transgenic mouse lines, and analyzed for the presence of GFP by Western blotting and fluorescence confirmed that the GUM promoter drove expression of GFP specifically in the kidney. More specifically, by using immuno-histochemistry analysis of kidney sections, we demonstrated that GFP expression was co-localized, with endogenous uromodulin protein, in the epithelial cells of the thick ascending limbs (TAL) of Henle's loop and the early distal convoluted tubule in the kidney. CONCLUSION: The goat uromodulin promoter is capable of driving recombinant protein expression in the kidney of transgenic mice. The goat promoter fragment cloned may be a useful tool in targeting proteins or oncogenes in the kidney of mammals

Substantially improved pharmacokinetics of recombinant human butyrylcholinesterase by fusion to human serum albumin

<p>Abstract</p> <p>Background</p> <p>Human butyrylcholinesterase (huBChE) has been shown to be an effective antidote against multiple LD₅₀of organophosphorus compounds. A prerequisite for such use of huBChE is a prolonged circulatory half-life. This study was undertaken to produce recombinant huBChE fused to human serum albumin (hSA) and characterize the fusion protein.</p> <p>Results</p> <p>Secretion level of the fusion protein produced <it>in vitro </it>in BHK cells was ~30 mg/liter. Transgenic mice and goats generated with the fusion constructs expressed in their milk a bioactive protein at concentrations of 0.04–1.1 g/liter. BChE activity gel staining and a size exclusion chromatography (SEC)-HPLC revealed that the fusion protein consisted of predominant dimers and some monomers. The protein was confirmed to have expected molecular mass of ~150 kDa by Western blot. The purified fusion protein produced <it>in vitro </it>was injected intravenously into juvenile pigs for pharmacokinetic study. Analysis of a series of blood samples using the Ellman assay revealed a substantial enhancement of the plasma half-life of the fusion protein (~32 h) when compared with a transgenically produced huBChE preparation containing >70% tetramer (~3 h). <it>In vitro </it>nerve agent binding and inhibition experiments indicated that the fusion protein in the milk of transgenic mice had similar inhibition characteristics compared to human plasma BChE against the nerve agents tested.</p> <p>Conclusion</p> <p>Both the pharmacokinetic study and the <it>in vitro </it>nerve agent binding and inhibition assay suggested that a fusion protein retaining both properties of huBChE and hSA is produced <it>in vitro </it>and <it>in vivo</it>. The production of the fusion protein in the milk of transgenic goats provided further evidence that sufficient quantities of BChE/hSA can be produced to serve as a cost-effective and reliable source of BChE for prophylaxis and post-exposure treatment.</p

International consensus guidelines for scoring the histopathological growth patterns of liver metastasis

BACKGROUND: Liver metastases present with distinct histopathological growth patterns (HGPs), including the desmoplastic, pushing and replacement HGPs and two rarer HGPs. The HGPs are defined owing to the distinct interface between the cancer cells and the adjacent normal liver parenchyma that is present in each pattern and can be scored from standard haematoxylin-and-eosin-stained (H&E) tissue sections. The current study provides consensus guidelines for scoring these HGPs. METHODS: Guidelines for defining the HGPs were established by a large international team. To assess the validity of these guidelines, 12 independent observers scored a set of 159 liver metastases and interobserver variability was measured. In an independent cohort of 374 patients with colorectal liver metastases (CRCLM), the impact of HGPs on overall survival after hepatectomy was determined. RESULTS: Good-to-excellent correlations (intraclass correlation coefficient >0.5) with the gold standard were obtained for the assessment of the replacement HGP and desmoplastic HGP. Overall survival was significantly superior in the desmoplastic HGP subgroup compared with the replacement or pushing HGP subgroup (P=0.006). CONCLUSIONS: The current guidelines allow for reproducible determination of liver metastasis HGPs. As HGPs impact overall survival after surgery for CRCLM, they may serve as a novel biomarker for individualised therapies

Eukaryotic translation initiation factor 4E : its characterization as a proto-oncogene and mechanism of action

Eukaryotic cellular mRNAs have a 5$sp prime$ cap structure (m7GpppX) that facilitates their binding to ribosomes and is required for efficient translation. An initiation factor, elF-4F, mediates the function of the cap and consists of three subunits. elF-4E, one of the subunits which binds the cap directly, is present in the cell in limiting amounts. We demonstrated that overexpression of elF-4E in NIH 3T3 and Rat 2 fibroblasts causes their tumorigenic transformation. We have also shown that elF-4E cooperates with an immortalizing, nuclear oncogene (ie. v-myc or E1A) to cause transformation of rat embryo fibroblasts. These findings categorize elF-4E as a proto-oncogene. The mode of transformation by elF-4E is similar to that of Ras-like proteins.To further elucidate the mechanism of elF-4E transformation we determined Ras activity in elF-4E transformed REFs. We detected an activation of Ras in eIF-4E overexpressing cells as compared to parental REFs. In addition, overexpression of GAP (GTPase activating-protein), a negative regulator of Ras, suppressed the transformation of REFs by elF-4E.In summary we have established an important link between Ras, which plays a key role in cellular signal transduction, and elF-4E which is a critical component of the translation machinery

Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3

Resistance to anti-angiogenic therapy is a major challenge in the treatment of colorectal cancer liver metastases (CRCLMs). Vessel co-option has been identified as a key contributor to anti-angiogenic therapy resistance in CRCLMs. Recently, we identified a positive correlation between the expression of Angiopoietin1 (Ang1) in the liver and the development of vessel co-opting CRCLM lesions in vivo. However, the mechanisms underlying its stimulation of vessel co-option are unclear. Herein, we demonstrated Ang1 as a positive regulator of actin-related protein 2/3 (ARP2/3) expression in cancer cells, in vitro and in vivo, which is known to be essential for the formation of vessel co-option in CRCLM. Significantly, Ang1-dependent ARP2/3 expression was impaired in the cancer cells upon Tie2 or PI3K/AKT inhibition in vitro. Taken together, our results suggest novel mechanisms by which Ang1 confers the development of vessel co-option in CRCLM, which, targeting this pathway, may serve as promising therapeutic targets to overcome the development of vessel co-option in CRCLM

Cancer Cells Promote Phenotypic Alterations in Hepatocytes at the Edge of Cancer Cell Nests to Facilitate Vessel Co-Option Establishment in Colorectal Cancer Liver Metastases

Vessel co-option is correlated with resistance against anti-angiogenic therapy in colorectal cancer liver metastases (CRCLM). Vessel co-opting lesions are characterized by highly motile cancer cells that move toward and along the pre-existing vessels in the surrounding nonmalignant tissue and co-opt them to gain access to nutrients. To access the sinusoidal vessels, the cancer cells in vessel co-opting lesions must displace the hepatocytes and occupy their space. However, the mechanisms underlying this displacement are unknown. Herein, we examined the involvement of apoptosis, autophagy, motility, and epithelial–mesenchymal transition (EMT) pathways in hepatocyte displacement by cancer cells. We demonstrate that cancer cells induce the expression of the proteins that are associated with the upregulation of apoptosis, motility, and EMT in adjacent hepatocytes in vitro and in vivo. Accordingly, we observe the upregulation of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and actin-related protein 2/3 (ARP2/3) in adjacent hepatocytes to cancer cell nests, while we notice a downregulation of E-cadherin. Importantly, the knockdown of runt-related transcription factor 1 (RUNX1) in cancer cells attenuates the function of cancer cells in hepatocytes alterations in vitro and in vivo. Altogether, our data suggest that cancer cells exploit various mechanisms to displace hepatocytes and access the sinusoidal vessels to establish vessel co-option

Histology-Driven Data Mining of Lipid Signatures from Multiple Imaging Mass Spectrometry Analyses: Application to Human Colorectal Cancer Liver Metastasis Biopsies

Imaging mass spectrometry (IMS) represents an innovative tool in the cancer research pipeline, which is increasingly being used in clinical and pharmaceutical applications. The unique properties of the technique, especially the amount of data generated, make the handling of data from multiple IMS acquisitions challenging. This work presents a histology-driven IMS approach aiming to identify discriminant lipid signatures from the simultaneous mining of IMS data sets from multiple samples. The feasibility of the developed workflow is evaluated on a set of three human colorectal cancer liver metastasis (CRCLM) tissue sections. Lipid IMS on tissue sections was performed using MALDI-TOF/TOF MS in both negative and positive ionization modes after 1,5-diaminonaphthalene matrix deposition by sublimation. The combination of both positive and negative acquisition results was performed during data mining to simplify the process and interrogate a larger lipidome into a single analysis. To reduce the complexity of the IMS data sets, a sub data set was generated by randomly selecting a fixed number of spectra from a histologically defined region of interest, resulting in a 10-fold data reduction. Principal component analysis confirmed that the molecular selectivity of the regions of interest is maintained after data reduction. Partial least-squares and heat map analyses demonstrated a selective signature of the CRCLM, revealing lipids that are significantly up- and down-regulated in the tumor region. This comprehensive approach is thus of interest for defining disease signatures directly from IMS data sets by the use of combinatory data mining, opening novel routes of investigation for addressing the demands of the clinical setting

Tissue extracts from a transgenic mouse (99-120-2F) and a negative control mouse (99-120-6F) were separated on 4–20 % SDS-PAGE and transferred onto the membrane

<p><b>Copyright information:</b></p><p>Taken from "Goat uromodulin promoter directs kidney-specific expression of GFP gene in transgenic mice"</p><p>BMC Biotechnology 2005;5():9-9.</p><p>Published online 11 Apr 2005</p><p>PMCID:PMC1090560.</p><p>Copyright © 2005 Huang et al; licensee BioMed Central Ltd.</p> The GFP positive control was from a cell line with GFP expression. 12 μg of total protein was loaded in each lane. Immunoreacting bands were detected using a polyclonal anti-GFP antibody (Clontech). . The same blot was reprobed with a mouse monoclonal anti-actin antibody (Chemicon) to show that equal amount of protein was loaded in each lane

Kidney extracts (12 μg of total protein loaded in each lane) from 99-120-2 and 99-122-1A3 were used as positive controls, whereas 99-120-6 was used as a negative control

<p><b>Copyright information:</b></p><p>Taken from "Goat uromodulin promoter directs kidney-specific expression of GFP gene in transgenic mice"</p><p>BMC Biotechnology 2005;5():9-9.</p><p>Published online 11 Apr 2005</p><p>PMCID:PMC1090560.</p><p>Copyright © 2005 Huang et al; licensee BioMed Central Ltd.</p> GFP immunoreacting protein was detected using the polyclonal anti-GFP antibody (Clontech) in 2 out 4 Day 0 pups, offspring of a transgenic F2 mouse, 00-074-1A2B8M, bred with a wild type mouse