Ornithine-δ-Aminotransferase Inhibits Neurogenesis During Xenopus Embryonic Development.

PURPOSE: In humans, deficiency of ornithine-δ-aminotransferase (OAT) results in progressive degeneration of the neural retina (gyrate atrophy) with blindness in the fourth decade. In this study, we used the Xenopus embryonic developmental model to study functions of the OAT gene on embryonic development. METHODS: We cloned and sequenced full-length OAT cDNA from Xenopus oocytes (X-OAT) and determined X-OAT expression in various developmental stages of Xenopus embryos and in a variety of adult tissues. The phenotype, gene expression of neural developmental markers, and enzymatic activity were detected by gain-of-function and loss-of-function manipulations. RESULTS: We showed that X-OAT is essential for Xenopus embryonic development, and overexpression of X-OAT produces a ventralized phenotype characterized by a small head, lack of axial structure, and defective expression of neural developmental markers. Using X-OAT mutants based on mutations identified in humans, we found that substitution of both Arg 180 and Leu 402 abrogated both X-OAT enzymatic activity and ability to modulate the developmental phenotype. Neurogenesis is inhibited by X-OAT during Xenopus embryonic development. CONCLUSIONS: Neurogenesis is inhibited by X-OAT during Xenopus embryonic development, but it is essential for Xenopus embryonic development. The Arg 180 and Leu 402 are crucial for these effects of the OAT molecule in development.

Cloning, expression, and functional characterization of human cyclooxygenase-1 splicing variants: evidence for intron 1 retention.

Recently, a splicing variant of cyclooxygenase (COX)-1, arising via the retention of its intron 1, was identified in canine. It was called COX-3 and was reported to be differentially sensitive to inhibition by various nonsteroidal anti-inflammatory drugs (NSAIDs) as well as acetaminophen (Chandrasekharan et al., 2002). However, the existence of an orthologous splicing variant in human tissues has been questioned due to a reading frame shift and premature termination. In this study, we first confirmed the existence of intron 1-retained COX-1 in certain human tissues at both the mRNA and protein levels. Molecular biology studies revealed that three distinct COX-1 splicing variants exist in human tissues. The most prevalent of these variants, called COX-1b1, arises via retention of the entire 94 base pair (bp) of intron 1, leading to a shift in the reading frame and termination at bp 249. However, the other two variant types, called COX-1b2 and COX-1b3, retain entire intron 1, but they are missing a nucleotide in one of two different positions, thereby encoding predicted full-length and likely COX-active proteins. Functional studies revealed that the COX-1b2 is able to catalyze the synthesis of prostaglandin F2alpha from arachidonic acid with Km and Vmax values of 0.54 microM and 3.07 pmol/mg/min, respectively. However, no significant differential selectivity for inhibition by selected NSAIDs was observed. Accordingly, we conclude that intron 1-retained human COX-1 is not likely to be the therapeutic target of acetaminophen or a candidate of COX-3.

Validation of in vivo pharmacodynamic activity of a novel PDGF receptor tyrosine kinase inhibitor using immunohistochemistry and quantitative image analysis.

With the advent of agents directed against specific molecular targets in drug discovery, it has become imperative to show a compound's cellular impact on the intended biomolecule in vivo. The objective of the present study was to determine if we could develop an assay to validate the in vivo effects of a compound. Hence, we investigated the in vivo pharmacodynamic activity of JNJ-10198409, a relatively selective inhibitor of platelet-derived growth factor receptor tyrosine kinase (PDGF-RTK), in tumor tissues after administering the compound orally in a nude mouse xenograft model of human LoVo colon cancer. We developed a novel assay to quantify the in vivo anti-PDGF-RTK activity of the inhibitor in tumor tissue by determining the phosphorylation status of phospholipase Cgamma1 (PLCgamma1), a key downstream cellular molecule in the PDGF-RTK signaling cascade. We used two antibodies, one specific for the total (phosphorylated and unphosphorylated forms) PLCgamma1 (pan-PLCgamma1) and the other, specific for phosphorylated form of PLCgamma1 (ph-PLCgamma1) to immunohistochemically detect their expression in tumor tissues. Computer-assisted image analysis was then used to directly compare the ratio of ph-PLCgamma1 to pan-PLCgamma1 immunolabeling intensities in serial sections (5 mum) of tumors obtained from vehicle- and JNJ-10198409-treated tumor-bearing mice. Our data showed statistically significant, dose-dependent differences in the ph-PLC/pan-PLC ratio among the four treatment groups (vehicle, 25, 50, and 100 mg/kg b.i.d.). These results confirmed this compound's ability to suppress PDGF-RTK downstream signaling in tumor tissues in vivo. In addition to this specific application of this in vivo validation approach to those targets that use PLCgamma as a downstream signaling partner, these methods may also benefit other drug discovery targets.

Matrix metalloproteinase(s) mediate(s) NO-induced dissociation of beta-catenin from membrane bound E-cadherin and formation of nuclear beta-catenin/LEF-1 complex.

Modulation of the adenomatous polyposis coli (APC)-beta-catenin pathway by inflammatory mediators and extracellular matrix may be important in colon carcinogenesis. We have recently shown that nitric oxide (NO) induces the accumulation of cytosolic beta-catenin and subsequent formation of the nuclear beta-catenin/lymphocyte enhancing factor (LEF)-1 complex in conditionally immortalized young mouse colonic epithelial (YAMC) cells. In the present study, we explored the mechanism(s) through which NO exerts its effect on cytosolic beta-catenin accumulation and nuclear beta-catenin/LEF-1 complex formation. We found that NO-induced degradation of the membrane bound E-cadherin at tight junctions. Using an anti-E-cadherin antibody specific for its extracellular domain, we detected a 50kDa degradation fragment of E-cadherin (120 kDa) from the culture medium conditioned by YAMC cells exposed to the NO-releasing drug, NOR-1, for 4 and 24 h. As beta-catenin is normally bound to transmembrane E-cadherin and thus anchored to the cytoskeleton structure, the degradation of E-cadherin induced by NO may cause dissociation of beta-catenin from membrane bound E-cadherin. This was demonstrated by the detection of beta-catenin accumulation in the soluble cytosolic fractions in YAMC after exposure to NO-releasing drugs. Furthermore, the degradation of E-cadherin and the release of beta-catenin to cytosol were accompanied by the formation of nuclear beta-catenin/LEF-1 complex, demonstrating the dissociation of beta-catenin from E-cadherin may be responsible for the activation of beta-catenin/LEF-1 transcription complex. Co-treatment with NO donors and broad-spectrum matrix metalloproteinase (MMP) inhibitors TIMP-1 (100 ng/ml), GM6001 (10 micro M) and GM1489 (10 micro M) abolished the degradation of E-cadherin induced by NO as demonstrated by western blot analysis. These MMP inhibitors also blocked the cytosolic accumulation of beta-catenin and nuclear formation of beta-catenin/LEF-1 complex. The sum effect of MMP inhibitors demonstrated that NO-induced activation of MMP may cause the degradation of E-cadherin and the subsequent dissociation of beta-catenin, thereby contributing to the cytosolic accumulation of beta-catenin and nuclear formation of beta-catenin/LEF-1 complex.