Genomic structure, alternative splicing and tissue expression of rFrp/sFRP-4, the rat frizzled related protein gene

Secreted frizzled related proteins (sFRP) are regulators of Wnt signaling pathways that play central roles in developmental processes and oncogenesis. Various sFRP genes have been cloned from different tissues and implicated in diverse biological activities. rFrp, the rat homologue of sFRP-4, was initially identified as being upregulated in mutant p53-induced cellular transformation. Here, we report on the isolation of five novel splice variants, rFrp/sFRP-4 II, II, III, IVa and IVb. The complete rFrp/sFRP-4 genomic structure spans over 31 kb covering 9 exons. Except for the variant IVb, which was derived from IVa by alternative polyadenylation signal, variants I to IVa were alternatively spliced to different exons in the 3'end of mRNA and resulted in transcripts with truncated open reading frame. The deduced proteins of the variants had truncated C-termini, however, the two key functional protein domains, the cysteine-rich domain and the netrin-like domain of the isoforms, were not altered. In addition, different transcriptional initiation sites were found with variants II and IV, implying that these variants may be regulated differently from the rFrp/sFRP-4. RT-PCR analysis showed that these splice variants displayed different patterns of tissue-specific expression. Northern blot analysis revealed that the rFrp/sFRP-4 is most abundant in the ovary. Taken together, our findings suggest that alternative splicing of rFrp/sFRP-4 plays a role in regulating tissue-specific expression. The truncated C terminals of rFrp/sFRP-4 variants may confer structural specificity and hence exert different biological functions in different tissues. Characterization of these novel splice variants should help to elucidate the function of the sFRP family gene. © 2005 Elsevier B.V. All rights reserved.postprin

Incorporating the ABI GeneScan® Analysis to a RACE-based technique for mapping multiple transcription initiation sites

Determination of transcription initiation sites has commonly been performed by primer extension and RNase protection assay using radioactively labeled oligonucleotides. Recently, a protocol based on modified 5′ rapid amplification of cDNA ends (RACE) with the use of fluorescently labeled primer was developed. Here, we describe the use of RACE-based technique in conjunction with the GeneScan® analysis for the determination of transcription initiation sites of genes of interest. The RACE technique is based on the ligation of an adapter to both ends of the cDNAs. The gene of interest was first amplified by PCR using a gene-specific and a 5′ adapter primer. Subsequently, nested PCR was performed using an internal gene-specific primer paired with a fluorescently end-labeled adapter primer. The size of the fluorescently labeled PCR products was directly determined by the ABI PRISM 377 GeneScan® Analyzer. This novel approach provides an accurate, sensitive, and convenient method for mapping transcription initiation sites, especially for genes with multiple transcription initiation sites, for genes expressed at low levels, and for splice variants that display alternative splicing farther than a few hundred nucleotides downstream from the transcription initiation site. This article describes the application of this new method in the mapping of transcription initiation sites of two splice variants of rat frizzled related protein transcripts.link_to_subscribed_fulltex

Characterization transgenic mice with the overexpression of ET-1 in endothelial cells as a model for experimental ischemic stroke

Endothelin-1 (ET-1), a potent vasoconstrictor, has been suggested to play a crucial role in cerebral ischemia. Previously, we have demonstrated that the ET-1 expressions were induced in astrocytic and endothelial cell after ischemic condition. Furthermore, ET-1 protected primary cultured astrocytes from hypoxic/ischemic injury. However, transgenic mice with overexpression of ET-1 in astrocytes displayed more severe neurological deficit and increased infract volume suggesting high level of astrocytic ET-1 have neurotoxic effect on neurons. In order to further investigate the role of endothelial ET-1 on neurological deficit and infarct after cerebral ischemia in vivo, several transgenic mouse lines were generated by injecting the construct which include ET cDNA and SV40 polyA under tyrosin kinase receptor-specific for endothelial cell (Tie-1) promoter which was shown to target reporter gene to endothelial cells. Over-expression of transgene, ET-1, in TET mouse brain has been characterized by reverse-transcription PCR and quantified by ribonuclease protection assay. Furthermore, the site of ET-1 over-expression in TET mice was determined by in situ hybridization. Our present TET model will serve as an ideal model for studying the pathophysiological mechanism of ischemic stroke. Keywords: endothelin-1, ischemic stroke, middle cerebral artery occlusion, nitric oxide.link_to_subscribed_fulltex

Effect of serum amyloid A1 treatment on global gene expression in THP-1-derived macrophages

10.1007/s00011-011-0424-4Inflammation Research614391-398INRE

c-Met Inhibitor Synergizes with Tumor Necrosis Factor–Related Apoptosis-Induced Ligand to Induce Papillary Thyroid Carcinoma Cell Death

The Met receptor tyrosine kinase is overexpressed and/or activated in variety of human malignancies. Previously we have shown that c-Met is overexpressed in Middle Eastern papillary thyroid carcinoma (PTC) and significantly associated with an aggressive phenotype, but its role has not been fully elucidated in PTC. The aim of this study was to determine the functional link between the c-Met/AKT signaling pathway and death receptor 5 (DR5) in a large cohort of PTC in a tissue microarray format followed by functional studies using PTC cell lines and nude mice. Our data showed that high expressions of p-Met and DR5 were significantly associated with an aggressive phenotype of PTC and correlated with BRAF mutation. Treatment of PTC cell lines with PHA665752, an inhibitor of c-Met tyrosine kinase, inhibited cell proliferation and induced apoptosis via the mitochondrial pathway in PTC cell lines. PHA665752 treatment or expression of c-Met small interfering (si)RNA resulted in dephosphorylation of c-Met, AKT and its downstream effector molecules. Furthermore, PHA665752 treatment upregulated DR5 expression via generation of reactive oxygen species in PTC cell lines, and synergistically potentiated death receptor–induced apoptosis with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). Finally, cotreatment with PHA665752 and TRAIL caused more pronounced effects on PTC xenograft tumor growth in nude mice. Our data suggest that the c-Met/AKT pathway may be a potential target for therapeutic intervention for treatment of PTC refractory to conventionally therapeutic modalities