Carboxypeptidase E (CPE) inhibits the secretion and activity of Wnt3a.

The Wnt pathway has essential roles in cell proliferation, cell fate determination and tumorigenesis by regulating the expression of a wide range of target genes. As a core signaling cascade, the canonical Wnt pathway is regulated at different levels by numerous proteins. We have previously shown that carboxypeptidase E (CPE) is a novel regulator of the canonical Wnt signaling pathway. Here, we show that CPE and the Wnt3a ligand are co-secreted from cells. We show that although the C'-terminal Lys residue of Wnt3a is critical for its activity and is important for the effect of CPE on the Wnt pathway, CPE does not execute its effect by removing this Wnt3a residue. Interestingly, CPE through its N'-terminal sequence, forms aggregates with Wnt3a and possible endoplasmic reticulum (ER) stress leading to its loss of function. Together, our current results provide a mechanistic insight into the way CPE regulates the canonical Wnt signaling pathway.

Carboxypeptidase E: a negative regulator of the canonical Wnt signaling pathway.

Aberrant activation of the canonical Wnt signal transduction pathway is involved in many diseases including cancer and is especially implicated in the development and progression of colorectal cancer. The key effector protein of the canonical Wnt pathway is ß-catenin, which functions with T-cell factor/lymphoid enhancer factor to activate expression of Wnt target genes. In this study, we used a new functional screen based on cell survival in the presence of cDNAs encoding proteins that activate the Wnt pathway thus identifying novel Wnt signaling components. Here we identify carboxypeptidase E (|CPE) and its splice variant, ΔN-CPE, as novel regulators of the Wnt pathway. We show that whereas ΔN-CPE activates the Wnt signal, the full-length CPE (F-CPE) protein is an inhibitor of Wnt/ß-catenin signaling. F-CPE forms a complex with the Wnt3a ligand and the Frizzled receptor. Moreover, F-CPE disrupts disheveled-induced signalosomes that are important for transducing the Wnt signal and reduces ß-catenin protein levels and activity. Taken together, our data indicate that F-CPE and ΔN-CPE regulate the canonical Wnt signaling pathway negatively and positively, respectively, and demonstrate that this screening approach can be a rapid means for isolation of novel Wnt signaling components.

Nuclear GSK-3beta inhibits the canonical Wnt signalling pathway in a beta-catenin phosphorylation-independent manner.

Beta-catenin is the central signalling molecule of the canonical Wnt pathway, where it activates target genes in a complex with lymphoid enhancer factor/T-cell factor transcription factors in the nucleus. The regulation of beta-catenin activity is thought to occur via a cytoplasmatic multiprotein complex that includes the serine/threonine kinase glycogen synthase kinase-3beta (GSK-3beta) that phosphorylates beta-catenin, marking it for degradation by the proteasome. Here, we provide evidence showing that GSK-3beta has a nuclear function in downregulating the activity of beta-catenin. Using colorectal cell lines that express a mutant form of beta-catenin, which cannot be phosphorylated by GSK-3beta and ectopically expressed mutant beta-catenin protein, we show that nuclear GSK-3beta functions in a mechanism that does not involve beta-catenin phosphorylation to reduce the levels of Wnt signalling. We show that GSK-3beta enters the nucleus, forms a complex with beta-catenin and lowers the levels of beta-catenin/TCF-dependent transcription in a mechanism that involves GSK-3beta-Axin binding.

ngl9: a third MADS box gene expressed in alfalfa root nodules.

Expression of MADS box genes has previously been localized to the infected cells of alfalfa (Medicago sativa) root nodules. These genes represent the first putative transcription factors to be identified in nodules and are hypothesized to be involved in a signal transduction pathway initiated by the intracellular bacterium. The eventual activation of specific target genes defines pertinent characteristics of this nitrogen-fixing differentiated cell. In this study, we identify a third nodule MADS box gene, ngl9, and demonstrate that the DNA-binding activity of its protein product is dependent on the presence of a second MADS box protein, NMH7. Despite previous results to the contrary, both genes are expressed in the early stages of flower development, further strengthening the premise that nodule developmental programming may capitalize upon existing developmental cascades.

Interaction between LIS1 and doublecortin, two lissencephaly gene products.

Mutations in either LIS1 or DCX are the most common cause for type I lissencephaly. Here we report that LIS1 and DCX interact physically both in vitro and in vivo. Epitope-tagged DCX transiently expressed in COS cells can be co-immunoprecipitated with endogenous LIS1. Furthermore, endogenous DCX could be co-immunoprecipitated with endogenous LIS1 in embryonic brain extracts, demonstrating an in vivo association. The two protein products also co-localize in transfected cells and in primary neuronal cells. In addition, we demonstrate homodimerization of DCX in vitro. Using fragments of both LIS1 and DCX, the domains of interaction were mapped. LIS1 and DCX interact with tubulin and microtubules. Our results suggest that addition of DCX and LIS1 to tubulin enhances polymerization in an additive fashion. In in vitro competition assays, when LIS1 is added first, DCX competes with LIS1 in its binding to microtubules, but when DCX is added prior to the addition of LIS1 it enhances the binding of LIS1 to microtubules. We conclude that LIS1 and DCX cross-talk is important to microtubule function in the developing cerebral cortex.

Doublecortin mutations cluster in evolutionarily conserved functional domains.

Mutations in the X-linked gene doublecortin ( DCX ) result in lissencephaly in males or subcortical laminar heterotopia ('double cortex') in females. Various types of mutation were identified and the sequence differences included nonsense, splice site and missense mutations throughout the gene. Recently, we and others have demonstrated that DCX interacts and stabilizes microtubules. Here, we performed a detailed sequence analysis of DCX and DCX-like proteins from various organisms and defined an evolutionarily conserved Doublecortin (DC) domain. The domain typically appears in the N-terminus of proteins and consists of two tandemly repeated 80 amino acid regions. In the large majority of patients, missense mutations in DCX fall within the conserved regions. We hypothesized that these repeats may be important for microtubule binding. We expressed DCX or DCLK (KIAA0369) repeats in vitro and in vivo. Our results suggest that the first repeat binds tubulin but not microtubules and enhances microtubule polymerization. To study the functional consequences of DCX mutations, we overexpressed seven of the reported mutations in COS7 cells and examined their effect on the microtubule cytoskeleton. The results demonstrate that some of the mutations disrupt microtubules. The most severe effect was observed with a tyrosine to histidine mutation at amino acid 125 (Y125H). Produced as a recombinant protein, this mutation disrupts microtubules in vitro at high molar concentration. The positions of the different mutations are discussed according to the evolutionarily defined DC-repeat motif. The results from this study emphasize the importance of DCX-microtubule interaction during normal and abnormal brain development.

Homologs of the alpha- and beta-subunits of mammalian brain platelet-activating factor acetylhydrolase Ib in the Drosophila melanogaster genome.

The mammalian intracellular brain platelet-activating factor acetylhydrolase, implicated in the development of cerebral cortex, is a member of the phospholipase A2 superfamily. It is made up of a homodimer of the 45 kDa LIS1 protein (a product of the causative gene for type I lissencephaly) and a pair of homologous 26-kDa alpha-subunits which account for all the catalytic activity. LIS1 is hypothesized to regulate nuclear movement in migrating neurons through interactions with the cytoskeleton, while the alpha-subunits, whose structure is known, contain a trypsin-like triad within the framework of a unique tertiary fold. The physiological significance of the association of the two types of subunits is not known. In an effort to better understand the function of the complex we turned to genomic data mining in search of related proteins in lower eukaryotes. We found that the Drosophila melanogaster genome contains homologs of both alpha- and beta-subunits, and we cloned both genes. The alpha-subunit homolog has been overexpressed, purified and crystallized. It lacks two of the three active-site residues and, consequently, is catalytically inactive against PAF-AH (Ib) substrates. Our study shows that the beta-subunit homolog is highly conserved from Drosophila to mammals and is able to interact with the mammalian alpha-subunits but is unable to interact with the Drosophila alpha-subunit. Proteins 2000;39:1-8.

Doublecortin, a stabilizer of microtubules.

X-linked lissencephaly is a severe brain malformation affecting males. Recently it has been demonstrated that the doublecortin gene is implicated in this disorder. In order to study the function of Doublecortin, we analyzed the protein upon transfection of COS cells. Doublecortin was found to bind to the microtubule cytoskeleton. In vitro assays (using biochemical methods, DIC microscopy and electron microscopy) demonstrate that Doublecortin binds microtubules directly, stabilizes them and causes bundling. In vivo assays also show that Doublecortin stabilizes microtubules and causes bundling. Doublecortin is a basic protein with an iso-electric point of 10, typical of microtubule-binding proteins. However, its sequence contains no known microtubule-binding domain(s). The results obtained in this study with Doublecortin and our previous work on another lissencephaly gene ( LIS1 ) emphasize the central role of regulation of microtubule dynamics and stability during neuronal morphogenesis.

Evolutionary diversity of symbiotically induced nodule MADS box genes: characterization of nmhC5, a member of a novel subfamily.

Unique organs called nodules form on legume roots in response to intracellular infection by soil bacteria in the genus Rhizobium. This study describes a new MADS box gene, nmhC5, which along with nmh7 (J. Heard and K. Dunn, Proc. Natl. Acad. Sci. USA 92:5273-5277, 1995), is expressed in alfalfa (Medicago sativa) root nodules. Together, these genes represent the first putative transcription factors identified in nodules. Expression in a root-derived structure supports the growing sentiment that MADS box proteins have diverse roles in plant development. Comparison of the putative translation product of nmhC5 with those of other reported members of the MADS box family suggests that the overall structure of nmhC5 is conserved. Evolutionary analysis among the MADS box family showed that nmhC5 is orthologous to a root-expressed clone in Arabidopsis thaliana, agl17, and that nmh7 is similar to the floral subfamily with AP3 (DefA)/PI (Glo). Consistent with a prediction of homodimer formation, NMHC5 was shown to bind a CArG consensus sequence in vitro. In contrast, NMH7, which shows structural similarity to MADS box proteins that form heterodimers, did not bind the CArG element in an in vitro DNA-binding assay, suggesting the existence of an unknown dimer partner. The root-derived MADS box genes constitute a novel subfamily of vegetatively expressed MADS box genes. The evolutionary diversity between nmh7 and nmhC5 could represent an overall mechanistic conservation between plant developmental processes or could mean that nmh7 and nmhC5 make fundamentally different contributions to the development of the nodule.

A mobile intensive care ambulance--use by community primary care teams.

OBJECTIVE: This study investigates the use of a mobile intensive care ambulance (MICA) by primary care teams in a large, semi-rural health district in northern Israel. METHOD: We reviewed the MICA records covering three years for all calls from a doctor or a nurse working in a community primary care clinic. RESULTS: Over the period studied, there were 649 such calls. Two-thirds of the patients were male, 70.5% were aged 60+ and only 2% were children under the age of one year. Of the total, 358 (55.2%) calls originated from kibbutzim (communal settlements), although only 7.5% of the district's population lives on such settlements. There were 12 calls (1.9%) from Arab villages, where 27.5% of the district population resides. The MICA personnel were more intervention prone than was the primary care team. The service was utilized mainly for cardiovascular emergencies and, in this study, children and residents of Arab villages appear to have been underserved. There was a disproportionately high representation of elderly kibbutz members. CONCLUSIONS: There is a need for further research to assess standards for emergency equipment in primary care clinics, and continuous training of the primary care team. Introduction of a high service technology such as the MICA might be a problem in certain populations.

[Thrombolysis for evolving myocardial infarction in a rural primary care clinic].

Early thrombolytic therapy gives maximum benefit in acute myocardial infarction. In remote rural areas with no mobile intensive care service there is a significant delay between onset of symptoms and administration of thrombolytic therapy which has a critical impact on revascularization. Thrombolytic therapy with streptokinase 1,500,000 U was given in a primary care rural clinic to 2 patients with evolving myocardial infarction 45-50 minutes from onset of symptoms. In both patients, who were transported to hospital after the therapy, there were clinical signs of reperfusion. There were no complications during treatment or transportation. We conclude that thrombolytic therapy given for evolving myocardial infarction in a rural primary care clinic is possible and safe.

Effect of a pharmacist's and a nurse's interventions on cost of drug therapy in a medical intensive-care unit.

The effect of interventions by a pharmacist and a nurse on the cost of drug therapy in a medical intensive-care unit (ICU) was determined. A pharmacist taught cost-avoidance concepts related to medication use to a senior member of the nursing staff. The pharmacist and the nurse documented during a 91-day period all interventions that resulted in a discontinuation or change in drug therapy or involved nonformulary drug requests or serum drug concentration determinations. Costs that were avoided or added as a result of these interventions were determined using drug acquisition costs and (for interventions involving i.v. therapy) the cost of related supplies. Costs were calculated for both the initial 24-hour period after each intervention and the expected duration of therapy. The 345 interventions of the pharmacist and the nurse represented $6,383 in 24-hour cost avoidance and $23,993 in total cost avoidance. The majority of interventions resulted in discontinuation of medications or changes in drug dosage; most of those interventions involved antimicrobial agents. Other frequent interventions involved changes in route of administration and the interchange of therapeutic alternates. A pharmacist and a nurse had a positive impact on the cost of drug therapy in a medical ICU.