Two-dimensional oriented growth of Zn-MOF-on-Zr-MOF architecture: A highly sensitive and selective platform for detecting cancer markers.

Fabricating novel bimetallic metal organic framework (MOF) architectures and exploiting them as aptasensor scaffolds for detecting diverse analytes, especially cancer markers, have aroused widespread research attention. Herein, we report a novel strategy for obtaining ZnZr bimetallic MOFs via the MOF-on-MOF method and exploit them as an aptasensor platform for detecting the cancer marker protein tyrosine kinase-7 (PTK7). Basic characterizations reveal that the chemical structure, crystalline properties, and surface functionality of bimetallic ZnZr-MOFs can be modulated by changing the order of addition of metal precursors and organic ligands. The Zn-MOF-on-Zr-MOF hybrid exhibits a hierarchically decussated foliace, whereas Zr-MOF-on-Zn-MOF demonstrates a multilayered nanosheet structure. The electrochemical results reveal that Zr-MOF facilitates aptamer strand immobilization, whereas the Zn-MOF stabilizes the G-quadruplex formed by aptamer strands and PTK7. The Zn-MOF-on-Zr-MOF-based aptasensor outperforms the Zr-MOF-on-Zn-MOF-based one, providing ultralow detection limits of 0.84 and 0.66 pg mL-1, as obtained by electrochemical impedance spectroscopy and differential pulse voltammetry, respectively, within the PTK7 concentration range of 1.0 pg mL-1 to 1.0 ng mL-1. The proposed Zn-MOF-on-Zr-MOF-based aptasensor exhibits high selectivity in the presence of various interferences, good stability, reproducibility, and acceptability in human serum. The proposed strategy provides a new approach for fabricating ultrasensitive and selective bimetallic MOFs-based aptasensors and contributes to efforts to broaden their applications in early cancer diagnosis.

Identification of a biologically active fragment of ALK and LTK-Ligand 2 (augmentor-α).

Elucidating the physiological roles and modes of action of the recently discovered ligands (designated ALKAL1,2 or AUG-α,ß) of the receptor tyrosine kinases Anaplastic Lymphoma Kinase (ALK) and Leukocyte Tyrosine Kinase (LTK) has been limited by difficulties in producing sufficient amounts of the two ligands and their poor stability. Here we describe procedures for expression and purification of AUG-α and a deletion mutant lacking the N-terminal variable region. Detailed biochemical characterization of AUG-α by mass spectrometry shows that the four conserved cysteines located in the augmentor domain (AD) form two intramolecular disulfide bridges while a fifth, primate-specific cysteine located in the N-terminal variable region mediates dimerization through formation of a disulfide bridge between two AUG-α molecules. In contrast to AUG-α, the capacity of AUG-α AD to undergo dimerization is strongly compromised. However, full-length AUG-α and the AUG-α AD deletion mutant stimulate similar tyrosine phosphorylation of cells expressing either ALK or LTK. Both AUG-α and AUG-α AD also stimulate a similar profile of MAP kinase response in L6 cells and colony formation in soft agar by autocrine stimulation of NIH 3T3 cells expressing ALK. Moreover, both AUG-α and AUG-α AD stimulate neuronal differentiation of human neuroblastoma NB1 and PC12 cells in a similar dose-dependent manner. Taken together, these experiments show that deletion of the N-terminal variable region minimally affects the activity of AUG-α toward LTK or ALK stimulation in cultured cells. Reduced dimerization might be compensated by high local concentration of AUG-α AD bound to ALK at the cell membrane and by potential ligand-induced receptor-receptor interactions.

Anaplastic lymphoma kinase immunohistochemistry in lung adenocarcinomas: Evaluation of performance of standard manual method using D5F3 antibody.

OBJECTIVE: Immunohistochemistry (IHC) with anaplastic lymphoma kinase (ALK) antibodies is considered as an economical screening method in lung adenocarcinomas. Automated Ventana D5F3-IHC is approved by US Food and Drug Administration for targeted therapy; however, the automated IHC apparatus are not widely used in most laboratories. We evaluated the performance of ALK IHC using the manual semiquantitative method to assess the concordance with Ventana ALK IHC assay. MATERIALS AND METHODS: We tested 156 cases of primary lung adenocarcinomas for ALK protein expression by D5F3-IHC. The intensity of cytoplasmic staining was classified as 0 or 1+/2+/3+ (weak/medium/strong). Binary score of positive and negative was used for Ventana assay. A comparison analysis and clinicopathological features were recorded. RESULTS: ALK IHC was positive in 25 (16.02%) cases, of which 18 were men and mostly nonsmokers. The mean age for all patients was 55 years, and for ALK IHC-positive cases was 48 years. Nine of 25 (36%) ALK IHC-positive cases showed signet ring cell and mucinous morphology. On comparison, all, but one, cases positive by manual method showed positive results by automated assay. IHC negative cases by manual method were negative by Ventana assay. CONCLUSION: Manual IHC is equally effective in the detection of ALK-rearranged cases as automated methods. It can be easily integrated as a screening method into routine practice thus reducing the cost of automated systems. However, equivocal cases should be tested by approved methods.

Anaplastic Lymphoma Kinase Testing: IHC vs. FISH vs. NGS.

OPINION STATEMENT: Personalized targeted therapy has emerged as a promising strategy in lung cancer treatment, with current attention focused on elucidation and detection of oncogenic drivers responsible for tumor initiation and maintenance and development of drug resistance. In lung cancer, several oncogenic drivers have been reported, triggering the application of tyrosine kinase inhibitors (TKIs) to target these dysfunctional genes. The anaplastic lymphoma kinase (ALK) rearrangement is responsible for about 4-7% of all non-small cell lung cancers (NSCLCs) and perhaps as high as a third in specific patient populations such as younger, male, non-smokers with advanced stage, epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene (KRAS) wild type, and signet ring cell adenocarcinoma with abundant intracytoplasmic mucin. The selection of patients based on their ALK status is vital on account of the high response rates with the ALK-targeted agents in this subset of patients. Standardization and validation of ALK rearrangement detection methods is essential for accurate and reproducible results. There are currently three detection methods widely available in clinical practice, including fluorescent in situ hybridization (FISH), immunohistochemistry (IHC), and polymerase chain reaction (PCR)-based next generation sequencing (NGS) technology. However, the choice of diagnostic methodology for ALK rearrangement detection in clinical practice remains a matter of debate. With accumulating data enumerating the advantages and disadvantages of each of the three methods, combining more than one testing method for ALK fusion detection may be beneficial for patients. In this review, we will discuss the current methods used in ALK rearrangement detection with emphasis on their key advantages and disadvantages.

Modeling of Receptor Tyrosine Kinase Signaling: Computational and Experimental Protocols.

The advent of systems biology has convincingly demonstrated that the integration of experiments and dynamic modelling is a powerful approach to understand the cellular network biology. Here we present experimental and computational protocols that are necessary for applying this integrative approach to the quantitative studies of receptor tyrosine kinase (RTK) signaling networks. Signaling by RTKs controls multiple cellular processes, including the regulation of cell survival, motility, proliferation, differentiation, glucose metabolism, and apoptosis. We describe methods of model building and training on experimentally obtained quantitative datasets, as well as experimental methods of obtaining quantitative dose-response and temporal dependencies of protein phosphorylation and activities. The presented methods make possible (1) both the fine-grained modeling of complex signaling dynamics and identification of salient, course-grained network structures (such as feedback loops) that bring about intricate dynamics, and (2) experimental validation of dynamic models.

Dichotomous ALK-IHC Is a Better Predictor for ALK Inhibition Outcome than Traditional ALK-FISH in Advanced Non-Small Cell Lung Cancer.

Purpose: ALK rearrangement detection using FISH is the standard test to identify patients with non-small cell lung carcinoma (NSCLC) eligible for treatment with ALK inhibitors. Recently, ALK protein expression in resectable NSCLC showed predictive value. We evaluated tumor response rate and survival after crizotinib treatment of patients with advanced NSCLC with ALK activation using both dichotomous immunohistochemical (IHC) staining and FISH.Experimental Design: Patients with stage IV NSCLC treated with crizotinib were selected. Tumor response was assessed. ALK rearrangements were detected by FISH (Vysis ALK-break-apart FISH-Probe KIT) and IHC [Ventana ALK (D5F3) CDx assay]. Cohorts of patients with ALK-FISH-positive advanced NSCLC from four other hospitals were used for validation.Results: Twenty-nine consecutive patients with ALK-positive advanced NSCLC diagnosed by FISH and/or IHC on small biopsies or fine-needle aspirations (FNA) were treated with ALK inhibitors. All ALK-IHC-positive patients responded to crizotinib except three with primary resistance. No tumor response was observed in 13 ALK-FISH-positive but ALK-IHC-negative patients. This was confirmed in an external cohort of 16 patients. Receiver operator characteristic (ROC) curves for ALK-IHC and ALK-FISH compared with treatment outcome showed that dichotomous ALK-IHC outperforms ALK-FISH [tumor response area under the curve: (AUC), 0.86 vs. 0.64, P = 0.03; progression-free survival (PFS): AUC 0.86 vs. 0.36, P = 0.005; overall survival (OS): AUC, 0.78 vs. 0.41, P = 0.01, respectively].Conclusions: Dichotomous ALK-IHC is superior to ALK-FISH on small biopsies and FNA to predict tumor response and survival to crizotinib for patients with advanced NSCLC. Our data strongly suggest adapting the guidelines and using dichotomous ALK-IHC as standard companion diagnostic test to select patients with NSCLC who benefit from ALK-targeting therapy. Clin Cancer Res; 23(15); 4251-8. ©2017 AACR.

Cost-effectiveness of ceritinib in patients previously treated with crizotinib in anaplastic lymphoma kinase positive (ALK+) non-small cell lung cancer in Canada.

BACKGROUND: To assess the cost-effectiveness of ceritinib vs alternatives in patients who discontinue treatment with crizotinib in anaplastic lymphoma kinase-positive (ALK+) non-small cell lung cancer (NSCLC) from a Canadian public healthcare perspective. METHODS: A partitioned survival model with three health states (stable, progressive, and death) was developed. Comparators were chosen based on reported utilization from a retrospective Canadian chart study; comparators were pemetrexed, best supportive care (BSC), and historical control (HC). HC comprised of all treatment alternatives reported. Progression-free survival and overall survival for ceritinib were estimated using data reported from single-arm clinical trials (ASCEND-1 [NCT01283516] and ASCEND-2 [NCT01685060]). Survival data for comparators were obtained from published clinical trials in a NSCLC population and from a Canadian retrospective chart study. Parametric models were used to extrapolate outcomes beyond the trial period. Drug acquisition, administration, resource use, and adverse event (AE) costs were obtained from databases. Utilities for health states and disutilities for AEs based on EQ-5D were derived from literature. Incremental costs per quality-adjusted life year (QALY) gained were estimated. Univariate and probabilistic sensitivity analyses were performed. RESULTS: Over 4 years, ceritinib was associated with 0.86 QALYs and total direct costs of $89,740 for the post-ALK population. The incremental cost-effectiveness ratio (ICER) was $149,117 comparing ceritinib vs BSC, $80,100 vs pemetrexed, and $104,436 vs HC. Additional scenarios included comparison to docetaxel with an ICER of $149,780 and using utility scores reported from PROFILE 1007, with a reported ICER ranging from $67,311 vs pemetrexed to $119,926 vs BSC. Due to limitations in clinical efficacy input, extensive sensitivity analyses were carried out whereby results remained consistent with the base-case findings. CONCLUSION: Based on the willingness-to-pay threshold for end-of-life cancer drugs, ceritinib may be considered as a cost-effective option compared with other alternatives in patients who have progressed or are intolerant to crizotinib in Canada.

ALK translocation detection in non-small cell lung cancer cytological samples obtained by TBNA or EBUS-TBNA.

OBJECTIVE: As the diagnosis of non-small cell lung cancer (NSCLC) is based on cytology in around 70% of cases, it is important to use the same material for molecular analyses. Fluorescence in situ hybridization (FISH) is the only approved test for the detection of the translocation and inversion of anaplastic lymphoma kinase (ALK), but the optimal procedures for the fixation or staining of the sample before FISH evaluation have not been established. We investigated whether ALK gene status determined by FISH in a prospectively enrolled case series of patients was affected by fixation and staining. METHODS: One hundred and fifteen cytological samples were obtained by transbronchial needle aspiration (TBNA) or endobronchial ultrasound (EBUS)-TBNA from 109 patients with NSCLC. All samples were evaluated for epidermal growth factor receptor (EGFR) mutation by pyrosequencing and for ALK rearrangement by FISH. Specimens for ALK determination had been fixed with Cytofix(®) and/or Carnoy's solution or 10% formalin (cell blocks) and variously stained. RESULTS: Sixteen (14%) of the 115 samples were mutated for EGFR and 99 (86%) showed wild-type EGFR status. Of these 115 samples, 79 (69%) were negative for echinoderm microtubule-associated protein like 4 (EML4)-ALK translocation, nine (8%) were positive and 27 (23%) were unevaluable. In particular, 19 (26%) of the 72 Papanicolaou-stained smears fixed with Cytofix were unevaluable because of inadequate samples or cell overlapping; neither of the two May-Grünwald-Giemsa-stained samples were evaluable. Ten of 17 smears used for rapid on-site evaluation (ROSE) and immediately post-fixed in Carnoy's solution or 80% alcohol were evaluable. CONCLUSIONS: In this series, smears were unevaluable as a result of inadequate samples, cell overlapping or lack of fixation performed immediately after FNA.

PTMScan direct: identification and quantification of peptides from critical signaling proteins by immunoaffinity enrichment coupled with LC-MS/MS.

Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in signaling in a wide array of experimental systems, combining the specificity of traditional biochemical methods with the high number of data points and dynamic range of proteomic methods.

Separation and determination of peptide hormones by capillary electrophoresis with laser-induced fluorescence coupled with transient pseudo-isotachophoresis preconcentration.

A new method for the determination of the peptide hormones and their fragments by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection and transient pseudo-isotachophoresis (pseudo-tITP) preconcentration was established in this study. The LIF detector used an argon ion laser with excitation wavelength at 488 nm and emission wavelength at 535 nm. Fluorescein isothiocyanate (FITC) was used as precolumn derivatization reagent to label cholecystokinin tetrapeptide (CCK-4), neurotensin (NT), neurotensin hexapeptide (NT(8-13)), and neurokinin B (NKB). Borate (10 mmol/L, pH 9.0) was selected as derivatization medium to get the high efficiency. When the addition of 70% (v/v) methanol and 1% (m/v) sodium chloride (NaCl) to the sample matrix, and with borate buffer (110 mM, pH 9.5) and 20% (v/v) methanol as running buffer, a preconcentration based on the pseudo-tITP afforded 100-fold improvement in peak heights compared with the traditional hydrodynamic injection (2.3% capillary volume). The detection limits (signal/noise=3) based on peak height were found to be 0.04, 0.1, 0.2, and 0.08 nmol/L for NT(8-13), NT, NKB, and CCK-4, respectively. The method was validated and applied to qualitative analysis of NT and NT(8-13) in human cerebrospinal fluid sample.

Expression of discoidin domain receptor 2 (DDR2) extracellular domain in pichia pastoris and functional analysis in synovial fibroblasts and NIT3T3 cells.

Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, identified as a ligand for DDR2, up-regulates matrix metallloproteinase 1 (MMP-1) and MMP-2 expression in cellular matrix. To investigate the roles of DDR2 in destruction of cartilage in rheumatoid arthritis (RA) and tumor metastasis, we tried to express extracellular domain of DDR2 fused with a His tag to increase protein solubility and facilitate purification (without signal peptide and transmembrane domain, designated DR) in Pichia pastoris, purify the expressed protein, and characterize its function, for purpose of future application as a specific DDR2 antagonist. Two clones of relative high expression of His-DR were obtained. After purification by a Ni-NTA (nitric-tri-acetic acid) chromatographic column, soluble fused His-DR over 90% purity were obtained. Competitive binding inhibition assay demonstrated that expressed His-DR could block the binding of DDR2 and natural DDR2 receptors on NIT3T3 and synovial cell surfaces. Results of RT-PCR, Western blotting, and gelatinase zymography showed that His-DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIT3T3 cells and RA synoviocytes stimulated by collagen II. For MMP-1, the inhibitory effect was displayed at the levels of mRNA and protein, whereas for MMP-2 it was demonstrated at the level of protein physiological activity. All these findings suggested that the fused expressed His-DR inhibited the activity of natural DDR2, and relevant MMP-1 and MMP-2 expression in synoviocytes and NIH3T3 cells provoked by collagen II.

Identification of agrinSN isoform and muscle-specific receptor tyrosine kinase (MuSK) [corrected] in sperm.

We previously demonstrated several nicotinic acetylcholine receptor (nAChR) subunits and associated proteins in human sperm. Here, we identified in sperm for the first time two additional nAChR-associated molecules: (1) agrin(SN)Z(+) in human sperm localized in the posterior post-acrosomal, neck, and flagellar mid-piece regions; (2) a low-molecular weight isoform of muscle-specific receptor tyrosine kinase in human and mouse sperm localized in the flagellar mid-piece of human sperm.

Expression, purification, and inhibition of human RET tyrosine kinase.

Tyrosine kinases are emerging as frequent targets of primary oncogenic events and therefore represent an optimal focus of therapeutical intervention. Genetic alterations that cause dysregulated activation of the RET tyrosine kinase are responsible for a significant fraction of thyroid carcinomas. In an effort towards therapeutic RET inactivation, we have developed a method for expression and purification of recombinant RET catalytic domain for structural purposes and for use in the screening of potential inhibitors of RET kinase activity. His-tagged RET kinase domain was purified from Sf9 insect cell lysate using a two-step chromatographic protocol and characterised. Purified recombinant RET phosphorylated itself and exogenous substrates at physiological pH. A specific peptide substrate, derived from RET activation loop, was identified and experimentally validated. These reagents were used to develop a rapid ELISA-based kinase assay for screening potential inhibitors. Novel RET inhibitors were identified using this assay.

C. elegans LIN-18 is a Ryk ortholog and functions in parallel to LIN-17/Frizzled in Wnt signaling.

Wnt proteins are intercellular signals that regulate various aspects of animal development. In Caenorhabditis elegans, mutations in lin-17, a Frizzled-class Wnt receptor, and in lin-18 affect cell fate patterning in the P7.p vulval lineage. We found that lin-18 encodes a member of the Ryk/Derailed family of tyrosine kinase-related receptors, recently found to function as Wnt receptors. Members of this family have nonactive kinase domains. The LIN-18 kinase domain is dispensable for LIN-18 function, while the Wnt binding WIF domain is required. We also found that Wnt proteins LIN-44, MOM-2, and CWN-2 redundantly regulate P7.p patterning. Genetic interactions indicate that LIN-17 and LIN-18 function independently of each other in parallel pathways, and different ligands display different receptor specificities. Thus, two independent Wnt signaling pathways, one employing a Ryk receptor and the other a Frizzled receptor, function in parallel to regulate cell fate patterning in the C. elegans vulva.

Characterization of a single-chain intrabody directed against the human receptor tyrosine kinase Ron.

A large human nonimmune phage antibody library was screened by affinity chromatography to select single-chain antibodies directed against the human receptor tyrosine kinase (RTK) Ron. As antigen, we used a GST fusion protein (GST-IRP(-)) containing the whole intracellular portion of Ron except for the carboxyl-terminal arginine-proline-rich motif. One selected phage was highly specific for Ron when tested in an enzyme-linked immunosorbent assay (ELISA). We report here the immunological characterization of this anti-Ron single-chain antibody (sc7) and show that it recognizes both denatured and native forms of the receptor. The epitope bound by sc7 maps within the first 50 amino acid residues of the juxtamembrane domain of Ron. This monoclonal fragment does not cross-react with other receptor tyrosine kinases including the closely related human proto-oncogene Met. We demonstrate that the isolated antibody fragment interacts in vivo with the intracellular domain of Ron in mammalian cells.

[Schistosoma mansoni receptor tyrosine kinases: towards new therapeutic targets]. / Les récepteurs tyrosine kinases de Schistosoma mansoni: à la recherche de nouvelles cibles thérapeutiques.

In spite of numerous efforts towards the control of its transmission, schistosomiasis still remains an important parasitic disease and represents a serious public health concern and a major economical problem in a lot of developing countries. The detection in different S. mansoni endemic areas of resistance to Praziquantel, the only drug currently used against the parasite, was sufficient to motivate actively further research for the discovery of novel drug treatments. Specific inhibitors for tyrosine kinase receptors (such as EGF receptor) are currently used with success as anti-tumor drugs. As cell proliferation and differentiation are essential events in the complex life cycle of the schistosome, we have attempted to consider parasite growth factor receptors as potential targets for a new generation of anti-parasitic agents. Three RTK have been identified in S. mansoni: an EGF receptor, an insulin receptor and a third receptor with an original structure probably belonging to a new class of RTK never identified. Structural and functional analyses of the parasite receptors demonstrated the conservation but also the divergences with their vertebrate counterparts, which are therefore excellent candidates for strategies of specific parasite RTK inhibition.

Identification of GFR alpha-2 isoforms in myenteric plexus of postnatal and adult rat intestine.

Glial cell line-derived neurotrophic factor family receptor alpha-2 (GFR alpha-2) is a GPI-linked receptor that preferentially binds neurturin (NTN), a member of the glial cell line-derived neurotrophic factor (GDNF) family. Three splice isoforms of GFR alpha-2 have been identified previously in mouse tissues, but the occurrence of splice isoforms in rats has not been described. The aim of this study was therefore to identify GFR alpha-2 splice isoforms in rat tissues using reverse transcription-polymerase chain reaction (RT-PCR) and gene cloning. Three isoforms were identified and sequenced, and named GFR alpha-2(a), (b) and (c), according to the nomenclature used for the previously identified mouse isoforms. The GFR alpha-2(a) and (b) isoforms were identical to those previously described in mice. The GFR alpha-2(c) isoform was novel. Sequences for GFR alpha-2(b) and (c) were deposited in the GenBank database (accession numbers GI: 16797788 and 16797786, respectively). All three isoforms were expressed in the brain, kidney, and intestine of both postnatal and adult rats.

Identification of functional VEGF receptors on human platelets.

Platelets secrete platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) upon stimulation. We have demonstrated that platelets have functionally active PDGF alpha-receptors, a transmembrane tyrosine kinase involved in negative feedback regulation. Here we demonstrate the presence of the related VEGF receptors fms-like tyrosine kinase-1 and kinase-insert domain region on human platelets. VEGF itself did not cause platelet aggregation. However, addition of exogenous VEGF to SFRLLN or thrombin-stimulated platelets potentiated platelet aggregation. Moreover, thrombin-induced phosphoinositide 3-kinase and mitogen-activated protein kinase activity were enhanced in the presence of VEGF.

Quantification of mouse glial cell-line derived neurotrophic factor family receptor alpha 2 alternatively spliced isoforms by real time detection PCR using SYBR Green I.

Neurturin (NTN) belongs to the glial cell-line derived neurotrophic factor (GDNF) family of growth factors. Both NTN and GDNF have been shown to potently prevent the degeneration of dopaminergic neuron in vitro and in vivo. The GDNF family receptor alpha 2 (GFR alpha-2) is the preferred receptor for NTN. In addition to the known full-length isoform (GFR alpha-2a), we have previously reported the isolation of two novel alternatively spliced isoforms (GFR alpha-2b and GFR alpha-2c). All three isoforms are expressed in all mammalian tissues examined, including human fetal brain. However, the expression levels of these isoforms have yet to be quantified. In this report, we have developed a real time polymerase chain reaction (PCR) detection method using SYBR Green I to detect the expression levels of the three splice variants (GFR alpha-2a, GFR alpha-2b and GFR alpha-2c). Of the three isoforms, GFR alpha-2a was found to be the most abundant receptor expressed in the whole murine brain. The real time PCR detection method using SYBR Green I developed in this report can be used to unambiguously quantitate expression levels of the GFR alpha-2 isoforms and can be extended to the quantitation of other alternatively spliced isoforms.

Vax2 inactivation in mouse determines alteration of the eye dorsal-ventral axis, misrouting of the optic fibres and eye coloboma.

Vax2 is a homeobox gene whose expression is confined to the ventral region of the prospective neural retina. Overexpression of this gene at early stages of development in Xenopus and in chicken embryos determines a ventralisation of the retina, thus suggesting its role in the molecular pathway that underlies eye development. We describe the generation and characterisation of a mouse with a targeted null mutation of the Vax2 gene. Vax2 homozygous mutant mice display incomplete closure of the optic fissure that leads to eye coloboma. This phenotype is not fully penetrant, suggesting that additional factors contribute to its generation. Vax2 inactivation determines dorsalisation of the expression of mid-late (Ephb2 and Efnb2) but not early (Pax2 and Tbx5) markers of dorsal-ventral polarity in the developing retina. Finally, Vax2 mutant mice exhibit abnormal projections of ventral retinal ganglion cells. In particular, we observed the almost complete absence of ipsilaterally projecting retinal ganglion cells axons in the optic chiasm and alteration of the retinocollicular projections. All these findings indicate that Vax2 is required for the proper closure of the optic fissure, for the establishment of a physiological asymmetry on the dorsal-ventral axis of the eye and for the formation of appropriate retinocollicular connections.