Key Specifications Table
|Key Applications||Format||Host||Detection Methods|
|Description||LIGHT DIAGNOSTICS™ Rabies DFA III|
|Overview||FITC Conjugated blend of clones 502.2 & 103.7|
|Presentation||Liquid in phosphate-buffered solution with preservative (0.1% NaN3) and stabilizer protein.|
|Clone||103.7 & 502.2|
|Antibody Type||Monoclonal Antibody|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||When stored at 2° to 8°C, reagent is stable up to the expiration date printed on the label.|
|Material Size||5 mL|
|Reference overview||Pub Med ID|
|Visualizing the Replication Cycle of Bunyamwera Orthobunyavirus Expressing Fluorescent Protein-tagged Gc Glycoprotein.|
Shi X, van Mierlo J, French A, Elliott RM
J Virol 2010
The virion glycoproteins Gn and Gc of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family and also the Orthobunyavirus genus, are encoded by the M RNA genome segment, and are involved in both viral attachment and entry. After their synthesis Gn and Gc form a heterodimer in the ER and transport to the Golgi for virus assembly. The N-terminal half of the Gc ectodomain was previously shown to be dispensable for virus replication in cell culture (Shi, X., J. Goli, G. Clark, K. Brauburger, and R. M. Elliott. 2009. J. Gen. Virol. 90:2483-2492). In this study, the coding sequences for fluorescent proteins, either enhanced green fluorescent protein (eGFP) or mCherry fluorescent protein, were fused to the N-terminus of truncated Gc, and two recombinant BUNV (rBUNGc-eGFP and rBUNGc-mCherry) were rescued by reverse genetics. The recombinant viruses showed bright autofluorescence under UV light and were competent for replication in various mammalian cell lines. rBUNGc-mCherry was completely stable over 10 passages whereas internal, in-frame deletions occurred in the chimeric Gc-eGFP protein of rBUNGc-eGFP resulting in loss of fluorescence between passages 5 and 7. Autofluorescence of the recombinant viruses allowed visualization of different stages of the infection cycle, including virus attachment to the cell surface, budding of virus particles in Golgi membranes, and virus-induced morphological changes to the Golgi at later stages of infection. The fluorescent protein tagged viruses will be valuable reagents for live cell imaging studies to investigate virus entry, budding and morphogenesis in real-time.
|Molecular characterization of EG-VEGF-mediated angiogenesis: differential effects on microvascular and macrovascular endothelial cells.|
Brouillet S, Hoffmann P, Benharouga M, Salomon A, Schaal JP, Feige JJ, Alfaidy N
Mol Biol Cell 21 2832-43. Epub 2010 Jun 29. 2010
Endocrine gland derived vascular endothelial growth factor (EG-VEGF) also called prokineticin (PK1), has been identified and linked to several biological processes including angiogenesis. EG-VEGF is abundantly expressed in the highest vascularized organ, the human placenta. Here we characterized its angiogenic effect using different experimental procedures. Immunohistochemistry was used to localize EG-VEGF receptors (PROKR1 and PROKR2) in placental and umbilical cord tissue. Primary microvascular placental endothelial cell (HPEC) and umbilical vein-derived macrovascular EC (HUVEC) were used to assess its effects on proliferation, migration, cell survival, pseudovascular organization, spheroid sprouting, permeability and paracellular transport. siRNA and neutralizing antibody strategies were used to differentiate PROKR1- from PROKR2-mediated effects. Our results show that 1) HPEC and HUVEC express both types of receptors 2) EG-VEGF stimulates HPEC's proliferation, migration and survival, but increases only survival in HUVECs. and 3) EG-VEGF was more potent than VEGF in stimulating HPEC sprout formation, pseudovascular organization, and it significantly increases HPEC permeability and paracellular transport. More importantly, we demonstrated that PROKR1 mediates EG-VEGF angiogenic effects, whereas PROKR2 mediates cellular permeability. Altogether, these data characterized angiogenic processes mediated by EG-VEGF, depicted a new angiogenic factor in the placenta, and suggest a novel view of the regulation of angiogenesis in placental pathologies.Full Text Article
|Genome analysis of Moraxella catarrhalis strain RH4, a human respiratory tract pathogen.|
de Vries SP, van Hijum SA, Schueler W, Riesbeck K, Hays JP, Hermans PW, Bootsma HJ
J Bacteriol 192 3574-83. Epub 2010 May 7. 2010
Moraxella catarrhalis is an emerging human-restricted respiratory tract pathogen that is a common cause of childhood otitis media and exacerbations of chronic obstructive pulmonary disease in adults. Here, we report the first completely assembled and annotated genome sequence of an isolate of M. catarrhalis, strain RH4, which originally was isolated from blood of an infected patient. The RH4 genome consists of 1,863,286 nucleotides that form 1,886 protein-encoding genes. Comparison of the RH4 genome to the ATCC 43617 contigs demonstrated that the gene content of both strains is highly conserved. In silico phylogenetic analyses based on both 16S rRNA and multilocus sequence typing revealed that RH4 belongs to the seroresistant lineage. We were able to identify almost the entire repertoire of known M. catarrhalis virulence factors and mapped the members of the biosynthetic pathways for lipooligosaccharide, peptidoglycan, and type IV pili. Reconstruction of the central metabolic pathways suggested that RH4 relies on fatty acid and acetate metabolism, as the genes encoding the enzymes required for the glyoxylate pathway, the tricarboxylic acid cycle, the gluconeogenic pathway, the nonoxidative branch of the pentose phosphate pathway, the beta-oxidation pathway of fatty acids, and acetate metabolism were present. Moreover, pathways important for survival under challenging in vivo conditions, such as the iron-acquisition pathways, nitrogen metabolism, and oxidative stress responses, were identified. Finally, we showed by microarray expression profiling that approximately 88% of the predicted coding sequences are transcribed under in vitro conditions. Overall, these results provide a foundation for future research into the mechanisms of M. catarrhalis pathogenesis and vaccine development.Full Text Article
|Effects of the EGFR Inhibitor Erlotinib on Magnesium Handling.|
Dimke H, van der Wijst J, Alexander TR, Meijer IM, Mulder GM, Goor HV, Tejpar S, Hoenderop JG, Bindels RJ
J Am Soc Nephrol 2010
A mutation in pro-EGF causes isolated hypomagnesemia, and monoclonal antibodies targeting the extracellular domain of the EGF receptor (EGFR) affect epithelial Mg(2+) transport. The effect of the EGFR tyrosine kinase inhibitor erlotinib on Mg(2+) homeostasis, however, remains unknown. Here, we injected C57BL/6 mice with erlotinib for 23 days and observed a small but significant decrease in serum Mg(2+) concentrations at days 16 and 23, but the fractional excretion of Mg(2+) remained unchanged after 23 days. Semiquantitative immunohistochemical evaluation did not reveal detectable changes in renal expression of transient receptor potential melastatin 6 (TRPM6) protein, the channel that mediates Mg(2+) reabsorption. Patch clamp analysis in TRPM6-expressing cells demonstrated that 30 mu M erlotinib inhibited EGF-induced changes in TRPM6 current density and tyrosine phosphorylation of EGFR; 0.3 mu M erlotinib did not have these effects. Furthermore, 30 mu M erlotinib inhibited EGF-stimulated increases in the mobile fraction of endomembrane TRPM6 channels. In summary, erlotinib can influence Mg(2+) handling but its effect on the systemic Mg(2+) concentration seems less potent than that observed with antibody-based EGFR inhibitors. These data suggest that typical human dosages of erlotinib are unlikely to severely affect serum Mg(2+) concentrations.
|Gene expression profiles of single human mature oocytes in relation to age.|
Grøndahl ML, Yding Andersen C, Bogstad J, Nielsen FC, Meinertz H, Borup R
Hum Reprod 25 957-68. Epub 2010 Feb 10. 2010
BACKGROUND: The development competence of human oocytes declines with increasing age. The objective of this study was to investigate the effect of age on gene expression profile in mature human oocytes. METHODS: mRNA was isolated for whole genome gene expression microarray analysis from metaphase II (MII) oocytes donated by IVF or ICSI patients [10 women aged <36 years (younger) and five women aged 37-39 years (both inclusive) (older)] undergoing controlled ovarian stimulation. The oocytes were donated and prepared immediately after recovery from the follicle. RT-PCR on additional four younger and two older oocytes confirmed the array analysis. RESULTS: On the basis of 15 independent replicates of single MII oocytes, 7470 genes (10 428 transcripts) were identified as present in the MII oocytes. Of these, 342 genes showed a significantly different expression level between the two age groups; notably, genes annotated to be involved in cell cycle regulation, chromosome alignment (e.g. MAD2L1 binding protein), sister chromatid separation (e.g. separase), oxidative stress and ubiquitination. The top signaling network affected by age was \'cell cycle and organism development\' (e.g. SMAD2 and activin B1 receptor). CONCLUSION: There is a substantial difference between younger and older oocytes in the transcriptional level of genes involved in central biological functions of the oocytes, thus providing information on processes that may be associated with the ageing phenomenon and possibly contributing to decreased fertility.
|The heparin-binding domain of HB-EGF mediates localization to sites of cell-cell contact and prevents HB-EGF proteolytic release.|
Prince RN, Schreiter ER, Zou P, Wiley HS, Ting AY, Lee RT, Lauffenburger DA
J Cell Sci 123 2308-18. Epub 2010 Jun 8. 2010
Heparin-binding EGF-like growth factor (HB-EGF) is a ligand for EGF receptor (EGFR) and possesses the ability to signal in juxtacrine, autocrine and/or paracrine mode, with these alternatives being governed by the degree of proteolytic release of the ligand. Although the spatial range of diffusion of released HB-EGF is restricted by binding heparan-sulfate proteoglycans (HSPGs) in the extracellular matrix and/or cellular glycocalyx, ascertaining mechanisms governing non-released HB-EGF localization is also important for understanding its effects. We have employed a new method for independently tracking the localization of the extracellular EGF-like domain of HB-EGF and the cytoplasmic C-terminus. A striking observation was the absence of the HB-EGF transmembrane pro-form from the leading edge of COS-7 cells in a wound-closure assay; instead, this protein localized in regions of cell-cell contact. A battery of detailed experiments found that this localization derives from a trans interaction between extracellular HSPGs and the HB-EGF heparin-binding domain, and that disruption of this interaction leads to increased release of soluble ligand and a switch in cell phenotype from juxtacrine-induced growth inhibition to autocrine-induced proliferation. Our results indicate that extracellular HSPGs serve to sequester the transmembrane pro-form of HB-EGF at the point of cell-cell contact, and that this plays a role in governing the balance between juxtacrine versus autocrine and paracrine signaling.
|Homeostatic expansion of autoreactive immunoglobulin-secreting cells in the Rag2 mouse model of Omenn syndrome.|
Cassani B, Poliani PL, Marrella V, Schena F, Sauer AV, Ravanini M, Strina D, Busse CE, Regenass S, Wardemann H, Martini A, Facchetti F, van der Burg M, Rolink AG, Vezzoni P, Grassi F, Traggiai E, Villa A
J Exp Med 207 1525-40. Epub 2010 Jun 14. 2010
Hypomorphic RAG mutations, leading to limited V(D)J rearrangements, cause Omenn syndrome (OS), a peculiar severe combined immunodeficiency associated with autoimmune-like manifestations. Whether B cells play a role in OS pathogenesis is so far unexplored. Here we report the detection of plasma cells in lymphoid organs of OS patients, in which circulating B cells are undetectable. Hypomorphic Rag2(R229Q) knock-in mice, which recapitulate OS, revealed, beyond severe B cell developmental arrest, a normal or even enlarged compartment of immunoglobulin-secreting cells (ISC). The size of this ISC compartment correlated with increased expression of Blimp1 and Xbp1, and these ISC were sustained by elevated levels of T cell derived homeostatic and effector cytokines. The detection of high affinity pathogenic autoantibodies toward target organs indicated defaults in B cell selection and tolerance induction. We hypothesize that impaired B cell receptor (BCR) editing and a serum B cell activating factor (BAFF) abundance might contribute toward the development of a pathogenic B cell repertoire in hypomorphic Rag2(R229Q) knock-in mice. BAFF-R blockade reduced serum levels of nucleic acid-specific autoantibodies and significantly ameliorated inflammatory tissue damage. These findings highlight a role for B cells in OS pathogenesis.
|Topical application of valrubicin has a beneficial effect on developing skin tumors.|
Andersen SM, Rosada C, Dagnaes-Hansen F, Laugesen IG, de Darkó E, Dam TN, Stenderup K
Carcinogenesis 31 1483-90. Epub 2010 Jun 16. 2010
Valrubicin is a second generation anthracycline characterized by an excellent safety profile presenting no skin toxicity or necrosis upon contact. In its current liquid formulation (Valstar; Indevus Pharmaceuticals, Lexington, MA), it is approved solely for the treatment of bladder cancer. Recently, valrubicin was incorporated in a cream formulation rendering this drug available for topical application. The cytostatic property of valrubicin can, thus, be employed for treating hyperproliferative skin diseases as was recently described for psoriasis. In the present study, the effect of topical application of valrubicin was investigated in skin tumor development; we hypothesized that valrubicin may be employed in treating actinic keratosis, a hyperproliferative skin condition that may transform into malignancy. A two-stage chemical mouse skin carcinogenesis model that represents the multistage etiology of human skin cancer-from developing papillomas to squamous cell carcinoma (SCC) was used. Moreover, two human skin SCC cell lines: DJM-1 and HSC-1 were cultured, to further investigate the effect of valrubicin in vitro. Cell viability was assessed by adenosine triphosphate presence, proliferation as proliferative cell nuclear antigen expression and apoptosis as cytokeratin 18 cleavage, caspase activation, poly-adenosine diphosphate-ribose-polymerase cleavage and bax and bcl-2 regulation. Valrubicin significantly inhibited tumor formation in the mouse skin carcinogenesis model and significantly decreased cell viability of the cultured human skin SCC cells. In both mouse skin and SCC cells, proliferation was significantly decreased. Apoptosis was significantly increased in SCC cells but unchanged in the treated mouse skin at study completion. This study demonstrated that topical application of valrubicin has a beneficial effect in treating developing skin tumors.
|Femtomolar detection of the anthrax edema factor in human and animal plasma.|
Elodie Duriez, Pierre L Goossens, François Becher, Eric Ezan
Analytical chemistry 81 5935-41 2009
Edema factor (EF), a calmodulin-activated adenylyl cyclase, is a toxin which contributes to cutaneous and systemic anthrax. As a novel strategy to detect anthrax toxins in humans or animals infected by Bacillus anthracis, we have developed a sensitive enzymatic assay to be able to monitor functional EF in human and animal plasma. Samples containing EF are incubated in the presence of calmodulin and ATP, which is converted to cAMP. After oxidation and derivatization, cAMP is monitored by competitive enzyme immunoassay. Because of the high turnover of EF and the sensitivity of cAMP detection, EF can be detected at concentrations of 1 pg/mL (10 fM) in 4 h in plasma from humans or at 10 pg/mL in the plasma of various animal species using only a blood volume of 5 microL. The assay has good reproducibility with intra- and interday coefficients of variation in the range of 20% and is not subject to significant interindividual matrix effects. In an experimental study performed in mice infected with the Berne strain, we were able to detect EF in serum and ear tissues. This simple and robust combination of enzymatic reaction and enzyme immunoassay for the diagnosis of anthrax toxemia could prove useful in biological threat detection as well in research and clinical practice.
|Purification of Candida guilliermondii and Pichia ohmeri killer toxin as an active agent against Penicillium expansum.|
Alexandre Rodrigo Coelho, Masahico Tachi, Fernando Carlos Pagnocca, Gisele Maria Andrade Nobrega, Fernando Leite Hoffmann, Ken-Ichi Harada, Elisa Yoko Hirooka
Food additives contaminants. Part A, Chemistry, analysis, control, exposure risk assessment 26 73-81 2009
An antifungal assay with cell-free culture supernatant of Pichia ohmeri 158 and Candida guilliermondii P3 was tested against Penicillium expansum strain #2 at 25 degrees C by measuring hyphal length and percentage conidia germination. C. guilliermondii was more effective against P. expansum conidia germination (58.15% inhibition), while P. ohmeri showed higher inhibition of mycelial growth (66.17%), indicating a probable mechanism associated with killer activity. This killer toxin (molecular mass 3 kDa) was partially purified by normal phase HPLC, using TSKgel Amide-80 analytical and preparative columns. Compared with crude extract, the killer toxin eluted from the post analytical column significantly inhibited P. expansum:% inhibition rose from 42.16 to 90.93% (C. guilliermondii) and 39.32 to 91.12% (P. ohmeri) (p 0.05). The one-step purification process was adequate in isolating killer toxin from culture supernatant and also increased anti-Penicillium activity.
|LIGHT DIAGNOSTICS RABIES DFA III REAGENT|