Neuronal ceroid lipofuscinosis in a mallard duck (Anas platyrhynchos)

A 3-year-old male, hybrid mallard duck (Anas platyrhynchos) was presented with a 2-year history of progressive, ascending paresis and paralysis. On presentation, the bird was in sternal recumbency and displayed intermittent focal seizures and intention tremors. Proprioceptive deficits and absent withdrawal reflexes were observed in both pelvic limbs, wing extension was limited, and reflexes were diminished bilaterally. Other findings included emaciation and dehydration. Results of a complete blood count and plasma biochemical analysis revealed anemia, hypoproteinemia, hypoglycemia, and hyperuricemia. Radiographs were unremarkable and results of an Aspergillus antibody were weakly positive. The duck went into cardiopulmonary arrest and died approximately 1 hour after initiating treatment with intravenous crystalloid fluids. A postmortem diagnosis of neuronal ceroid lipofuscinosis (NCL) was made based on the presence of neuronal granular cytoplasmic material that was autofluorescent and stained with periodic acid-Schiff and Luxol fast blue. To our knowledge, this is only the second case report of NCL in an avian species and the first in waterfowl.     

Smoking cessation research in primary care treatment centers: the SCRIPT-MS project

Despite wide distribution of an evidence-based clinical practice guideline, the provision of treatment for tobacco use has been weak. The primary care setting is an ideal environment in which to implement the tobacco clinical practice guideline. It has been suggested that implementation of the guideline may be enhanced by adapting guideline recommendations into a stepped-care plus treatment-matching model; however, this model has yet to be tested. This article describes an ongoing investigation designed to evaluate (1) the feasibility of implementing this treatment model in a primary care environment, (2) efforts on identifying reasonable assessment methods, (3) primary care providers' use of assessment data, and (4) the relationships between predictors and outcomes under differing treatment "step" conditions in primary care settings. Six primary care clinics are participating in this study comparing usual care, brief clinical intervention, and enhanced clinical intervention conditions. The last of these conditions is hypothesized to produce the best and most cost-effective outcomes. Recruitment is expected to continue until January 2004, being 65% complete as of this writing. Follow-up contacts will continue until April 2005. Results should provide information that will contribute to the ongoing development of primary care-based tobacco intervention approaches.     

Genome-wide copy number profiling reveals molecular evolution from diagnosis to relapse in childhood acute lymphoblastic leukemia

The underlying pathways that lead to relapse in childhood acute lymphoblastic leukemia (ALL) are unknown. To comprehensively characterize the molecular evolution of relapsed childhood B-precursor ALL, we used human 500K single-nucleotide polymorphism arrays to identify somatic copy number alterations (CNAs) in 20 diagnosis/relapse pairs relative to germ line. We identified 758 CNAs, 66.4% of which were less than 1 Mb, and deletions outnumbered amplifications by approximately 2.5:1. Although CNAs persisting from diagnosis to relapse were observed in all 20 cases, 17 patients exhibited differential CNA patterns from diagnosis to relapse. Of the 396 CNAs observed in 20 relapse samples, only 69 (17.4%) were novel (absent in the matched diagnosis samples). EBF1 and IKZF1 deletions were particularly frequent in this relapsed ALL cohort (25.0% and 35.0%, respectively), suggesting their role in disease recurrence. In addition, we noted concordance in global gene expression and DNA copy number changes (P = 2.2 x 10(-16)). Finally, relapse-specific focal deletion of MSH6 and, consequently, reduced gene expression were found in 2 of 20 cases. In an independent cohort of children with ALL, reduced expression of MSH6 was associated with resistance to mercaptopurine and prednisone, thereby providing a plausible mechanism by which this acquired deletion contributes to drug resistance at relapse.     

Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs

Posttranslational knockdown of a specific protein is an attractive approach for examining its function within a system. Here we introduce phospho-dependent proteolysis targeting chimeras (phosphoPROTACs), a method to couple the conditional degradation of targeted proteins to the activation state of particular kinase-signaling pathways. We generated two phosphoPROTACs that couple the tyrosine phosphorylation sequences of either the nerve growth factor receptor, TrkA (tropomyosin receptor kinase A), or the neuregulin receptor, ErbB3 (erythroblastosis oncogene B3), with a peptide ligand for the E3 ubiquitin ligase von Hippel Lindau protein. These phosphoPROTACs recruit either the neurotrophic signaling effector fibroblast growth factor receptor substrate 2α or the survival-promoting phosphatidylinositol-3-kinase, respectively, to be ubiquitinated and degraded upon activation of specific receptor tyrosine kinases and phosphorylation of the phosphoPROTACs. We demonstrate the ability of these phosphoPROTACs to suppress the short- and long-term effects of their respective activating receptor tyrosine kinase pathways both in vitro and in vivo. In addition, we show that activation of phosphoPROTACs is entirely dependent on their kinase-mediated phosphorylation, as phenylalanine-containing null variants are inactive. Furthermore, stimulation of unrelated growth factor receptors does not induce target protein knockdown. Although comparable in efficiency to RNAi, this approach has the added advantage of providing a degree of temporal and dosing control as well as cell-type selectivity unavailable using nucleic acid-based strategies. By varying the autophosphorylation sequence of a phosphoPROTAC, it is conceivable that other receptor tyrosine kinase/effector pairings could be similarly exploited to achieve other biological effects.     

Microglial depletion and repopulation: a new era of regenerative medicine?

Microglia have multiple functions and phenotypes that can prevent or worsen neuropathology. Microglial depletion and repopulation methods provide a promising te...     

Receptor-mediated inositide hydrolysis is a neuronal response: comparison of primary neuronal and glial cultures

Cholinergic and adrenergic receptor-stimulated inositide hydrolysis was studied in neuronal and glial cells cultured from brains of 1-day-old Wistar-Kyoto rats. Incubation of the cells with [3H]inositol led to the incorporation of radioactivity specifically into inositol phospholipids. Labeling of the membrane lipids reached a maximum in 2-3 days. Receptor-stimulated breakdown of inositides was determined by following the accumulation of inositol phosphates after incubation of the labeled cells for 60 min with carbachol or norepinephrine in the presence of 10 mM lithium. Carbachol (1 mM) stimulated inositol phosphate production in neurons 30 times higher than that seen in glia. The response stimulated by norepinephrine (75 microM) was 6 times higher in neurons than glia. The response to carbachol was blocked by atropine, and the norepinephrine-induced response was inhibited by prazosin suggesting that the receptors mediating the responses were muscarinic and alpha 1-adrenergic, respectively. These results suggest that muscarinic cholinergic and alpha 1-adrenergic stimulated inositide hydrolysis is primarily a neuronal response and that this biochemical event may be important for transmembrane signaling which occurs during neurotransmission.