05-533 Sigma-AldrichAnti-phospho-CaM Kinase II Antibody, α subunit, (Thr286), clone 22B1

Signals emanating from the bone marrow microenvironment, such as stromal cells, are thought to support the survival and proliferation of the malignant cells in patients with myeloproliferative neoplasms (MPN). To examine this hypothesis, we established a coculture platform [cells cocultured directly (cell-on-cell) or indirectly (separated by micropore membrane)] designed to interrogate the interplay between Janus activated kinase 2-V617F (JAK2(V617F))-positive cells and the stromal cells. Treatment with atiprimod, a potent JAK2 inhibitor, caused marked growth inhibition and apoptosis of human (SET-2) and mouse (FDCP-EpoR) JAK2(V617F)-positive cells as well as primary blood or bone marrow mononuclear cells from patients with polycythemia vera; however, these effects were attenuated when any of these cell types were cocultured (cell-on-cell) with human marrow stromal cell lines (e.g., HS5, NK.tert, TM-R1). Coculture with stromal cells hampered the ability of atiprimod to inhibit phosphorylation of JAK2 and the downstream STAT3 and STAT5 pathways. This protective effect was maintained in noncontact coculture assays (JAK2(V617F)-positive cells separated by 0.4-μm-thick micropore membranes from stromal cells), indicating a paracrine effect. Cytokine profiling of supernatants from noncontact coculture assays detected distinctly high levels of interleukin 6 (IL-6), fibroblast growth factor (FGF), and chemokine C-X-C-motif ligand 10 (CXCL-10)/IFN-γ-inducible 10-kD protein (IP-10). Anti-IL-6, -FGF, or -CXCL-10/IP-10 neutralizing antibodies ablated the protective effect of stromal cells and restored atiprimod-induced apoptosis of JAK2(V617F)-positive cells. Therefore, our results indicate that humoral factors secreted by stromal cells protect MPN clones from JAK2 inhibitor therapy, thus underscoring the importance of targeting the marrow niche in MPN for therapeutic purposes.

Animal models of human diseases are critical for dissecting mechanisms of pathophysiology and developing therapies. In the context of cystic fibrosis (CF), mouse models have been the dominant species by which to study CF disease processes in vivo for the past two decades. Although much has been learned through these CF mouse models, limitations in the ability of this species to recapitulate spontaneous lung disease and several other organ abnormalities seen in CF humans have created a need for additional species on which to study CF. To this end, pig and ferret CF models have been generated by somatic cell nuclear transfer and are currently being characterized. These new larger animal models have phenotypes that appear to closely resemble human CF disease seen in newborns, and efforts to characterize their adult phenotypes are ongoing. This chapter will review current knowledge about comparative lung cell biology and cystic fibrosis transmembrane conductance regulator (CFTR) biology among mice, pigs, and ferrets that has implications for CF disease modeling in these species. We will focus on methods used to compare the biology and function of CFTR between these species and their relevance to phenotypes seen in the animal models. These cross-species comparisons and the development of both the pig and the ferret CF models may help elucidate pathophysiologic mechanisms of CF lung disease and lead to new therapeutic approaches.

Neurological deficits in the offspring caused by human maternal hypothyroxinemia are thought to be irreversible. To understand the mechanism responsible for these neurological alterations, we induced maternal hypothyroxinemia in pregnant rats. Behavior and synapse function were evaluated in the offspring of thyroid hormone-deficient rats. Our data indicate that, when compared with controls, hypothyroxinemic mothers bear litters that, in adulthood, show prolonged latencies during the learning process in the water maze test. Impaired learning capacity caused by hypothyroxinemia was consistent with cellular and molecular alterations, including: 1) lack of increase of phosphorylated c-fos on the second day of the water maze test; 2) impaired induction of long-term potentiation in response to theta-burst stimulation to the Schaffer collateral pathway in the area 1 of the hippocampus Ammon's horn stratum radiatum, despite normal responses for input/output experiments; 3) increase of postsynaptic density protein 95 (PSD-95), N-methyl-D-aspartic acid receptor subunit 1, and tyrosine receptor kinase B levels in brain extracts; and 4) significant increase of PSD-95 at the PSDs and failure of this molecule to colocalize with N-methyl-D-aspartic acid receptor subunit 1, as it was shown by control rats. Our findings suggest that maternal hypothyroxinemia is a harmful condition for the offspring that can affect key molecular components for synaptic function and spatial learning.

Excitatory stimuli are known to be a potent regulator for induction of neuronal differentiation. Calbindin-D28K buffers intracellular Ca2+ and modifies synaptic functions in neurons. However, the effects of calbindin-D28K on the regulation of activity-induced neuronal differentiation and related biochemical modifications remain unsolved. In the present study, by a gain-of-function study with retroviral vector system and dicer-generated small interfering RNA (d-siRNA) to effectively knock down the expression of calbindin-D28K, we demonstrated that calbindin-D28K at a physiologically relevant level promoted neuronal differentiation and neurite outgrowth. Increase of neuronal differentiation by calbindin-D28K overexpression was concurrent with the expression of basic helix-loop-helix (bHLH) transcriptional factors, phosphorylation of calcium and calmodulin-dependent protein kinase II (CaMKII) and NeuroD at Ser(336). KN-62, a highly specific CaMKII inhibitor, blocked the up-regulation of proneural bHLH genes, p-CaMKII, and pSer(336)NeuroD. Calbindin-D28K appeared to facilitate neuronal differentiation of both fetal and adult hippocampal progenitor cells. Together, these findings establish the novel calbindin-regulated function of CaMKII and NeuroD in control of neuronal differentiation and neurite outgrowth.

The present study provides the first evidence that adhesion receptors belonging to the integrin family modulate excitatory transmission in the adult rat brain. Infusion of an integrin ligand (the peptide GRGDSP) into rat hippocampal slices reversibly increased the slope and amplitude of excitatory postsynaptic potentials. This effect was not accompanied by changes in paired pulse facilitation, a test for perturbations to transmitter release, or affected by suppression of inhibitory responses, suggesting by exclusion that alterations to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors cause the enhanced responses. A mixture of function-blocking antibodies to integrin subunits alpha(3), alpha(5), and alpha(v) blocked ligand effects on synaptic responses. The ligand-induced increases were (i) blocked by inhibitors of Src tyrosine kinase, antagonists of N-methyl-d-aspartate receptors, and inhibitors of calcium calmodulin-dependent protein kinase II and (ii) accompanied by phosphorylation of both the Thr(286) site on calmodulin-dependent protein kinase II and the Ser(831) site on the GluR1 subunit of the AMPA receptor. N-Methyl-d-aspartate receptor antagonists blocked the latter two phosphorylation events, but Src kinase inhibitors did not. These results point to the conclusion that synaptic integrins regulate glutamatergic transmission and suggest that they do this by activating two signaling pathways directed at AMPA receptors.

We have visualized the distribution of autophosphorylated type II CaM kinase in neural tissue with the use of two complementary antibodies: a monoclonal antibody that binds to the alpha and beta subunits of the kinase only when they are autophosphorylated at threonine-286 (287 in beta) and affinity-purified rabbit antibodies that bind to both subunits only when they are not phosphorylated at these residues. We used these antibodies to double-label organotypic hippocampal cultures, detecting the mouse monoclonal antibody with rhodamine and the rabbit polyclonal antibodies with fluorescein. In double-exposed photographs, the ratios of intensities of the two fluorophores revealed the relative proportion of autophosphorylated and nonphosphorylated kinase in individual neurons throughout the cultures. We found that autophosphorylated and nonphosphorylated kinase are colocalized throughout most neurons rather than segregated within distinct cells or subcellular domains. However, the variations in intensity of the two fluorophores indicated that the proportion of autophosphorylated kinase is consistently higher in neuronal somas than in the neuropil. Incubation of the cultures in Ca2+ free medium dramatically reduced both the level of autophosphorylated kinase detected biochemically and the relative intensity of fluorescent staining with the phosphokinase specific monoclonal antibody. These results support the hypothesis that regulation of Ca(2+)-independent CaM kinase activity in vivo occurs by a dynamic equilibrium between autophosphorylation and dephosphorylation and that this equilibrium is maintained, at varying steady-state levels, in all parts of neurons.