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Alzheimer's disease (AD) is a common, chronic neurodegenerative disease that is thought to be caused by the neurotoxic effect of the Amyloid beta peptides (A[beta]). We have hypothesized that the intrinsic A[beta] calcium channel activity of the oligomeric A[beta] polymer may be responsible for the neurotoxic properties of A[beta], and that A[beta] channel blockers may be candidate AD therapeutics. As a consequence of a rational search paradigm based on the model structure of the A[beta] channel, we have identified two compounds of interest: MRS2481 and an enatiomeric species, MRS2485. These are amphiphilic pyridinium salts that both potently block the A[beta] channel and protect neurons from A[beta] toxicity. Both block the A[beta] channel with similar potency ([approximately equal to] 500 nM) and efficacy (100%). However, we find that inhibition by MRS2481 is easily reversible, whereas inhibition by MRS2485 is virtually irreversible. We suggest that both species deserve consideration as candidates for Alzheimer's disease drug discovery. brain | neurodegeneration | rational drug design | drug | toxicity

Digitoxin and other cardiac glycosides are important, centuries-old drugs for treating congestive heart failure. However, the mechanism of action of these compounds is still being elucidated. Calcium is known to potentiate the toxicity of these drugs, and we have hypothesized that digitoxin might mediate calcium entry into cells. We report here that digitoxin molecules mediate calcium entry into intact cells. Multimers of digitoxin molecules also are able to form calcium channels in pure planar phospholipid bilayers. These digitoxin channels are blocked by [Al.sup.3+] and [La.sup.3+] but not by [Mg.sup.2+]or the classical L-type calcium channel blocker, nitrendipine. In bilayers, we find that the chemistry of the lipid affects the kinetics of the digitoxin channel activity, but not the cation selectivity. Antibodies against digitoxin promptly neutralize digitoxin channels in both cells and bilayers. We propose that these digitoxin calcium channels may be part of the mechanism by which digitoxin and other active cardiac glycosides, such as digoxin, exert system-wide actions at and above the therapeutic concentration range. cardiac glycoside | cytotoxicity

Digitoxin and structurally related cardiac glycoside drugs potently block activation of the TNF-[alpha]/NF-[kappa]B signaling pathway. We have hypothesized that the mechanism might be discovered by searching systematically for selective inhibitory action through the entire pathway. We report that the common action of these drugs is to block the TNF-[alpha]-dependent binding of TNF receptor 1 to TNF receptor-associated death domain. This drug action can be observed with native cells, such as HeLa, and reconstituted systems prepared in HEK293 cells. All other antiinflammatory effects of digitoxin on NF-[kappa]B and c-Jun N-terminal kinase pathways appear to follow from the blockade of this initial upstream signaling event. digitoxin | inflammation

Cystic fibrosis (CF) is a fatal, autosomal, recessive genetic disease that is characterized by profound lung inflammation. The inflammatory process is believed to be caused by massive overproduction of the proinflammatory protein IL-8, and the high levels of IL-8 in the CF lung are therefore believed to be the central mechanism behind CF lung pathophysiology. We show here that digitoxin, at sub nM concentrations, can suppress hypersecretion of IL-8 from cultured CF lung epithelial cells. Certain other cardiac glycosides are also active but with much less potency. The specific mechanism of digitoxin action is to block phosphorylation of the inhibitor of NF-[kappa]B (I[kappa]B[alpha]). I[kappa]B[alpha] phosphorylation is a required step in the activation of the NF-[kappa]B signaling pathway and the subsequent expression of IL-8. Digitoxin also has effects on global gene expression in CF cells. Of the informative genes expressed by the CF epithelial cell line IB-3, 58 are significantly (P < 0.05) affected by gene therapy with wild-type (CFTR CF transmembrane conductance regulator). Of these 58 genes, 36 (62%) are similarly affected by digitoxin and related active analogues. We interpret this result to suggest that digitoxin can also partially mimic the genomic consequences of gene therapy with CF transmembrane conductance regulator. We therefore suggest that digitoxin, with its lengthy history of human use, deserves consideration as a candidate drug for suppressing IL-8-dependent lung inflammation in CF.

Annexin 7 (ANX7) acts as a tumor suppressor gene in prostate cancer, where loss of heterozygosity and reduction of ANX7 protein expression is associated with aggressive metastatic tumors. To investigate the mechanism by which this gene controls tumor development, we have developed an Anx7(+/-) knockout mouse. As hypothesized, the Anx7(+/-) mouse has a cancer-prone phenotype. The emerging tumors express low levels of Anx7 protein. Nonetheless, the wild-type Anx7 allele is detectable in laser-capture microdissection-derived tumor tissue cells. Genome array analysis of hepatocellular carcinoma tissue indicates that the Anx7(+/-) genotype is accompanied by profound reductions of expression of several other tumor suppressor genes, DNA repair genes, and apoptosis-related genes. In situ analysis by tissue imprinting from chromosomes in the primary tumor and spectral karyotyping analysis of derived cell lines identify chromosomal instability and clonal chromosomal aberrations. Furthermore, whereas 23% of the mutant mice develop spontaneous neoplasms, all mice exhibit growth anomalies, including gender-specific gigantism and organomegaly. We conclude that haploinsufficiency of Anx7 expression appears to drive disease progression to cancer because of genomic instability through a discrete signaling pathway involving other tumor suppressor genes, DNA-repair genes, and apoptosis-related genes. hepatocellular carcinoma lymphosarcoma of the thymus SKY analysis cDNA microarray

Exocytotic membrane fusion and secretion are promoted by the concerted action of GTP and [Ca.sup.2+], although the precise site(s) of action in the process are not presently known. However, the calcium-dependent membrane fusion reaction driven by synexin (annexin VII) is an in vitro model for this process, which we have now found to be further activated by GTP. The mechanism of fusion activation depends on the unique ability of synexin to bind and hydrolyze GTP in a calcium-dependent manner, both in vitro and in vivo in streptolysin O-permeabilized chromaffin cells. The required [[Ca.sup.2+]] for GTP binding by synexin is in the range of 50-200 [[micro]molar], which is known to occur at exocytotic sites in chromaffin cells, neurons, and other cell types. Previous immunolocalization studies place synexin at exocytotic sites in chromaffin cells, and we conclude that synexin is an atypical G protein that may be responsible for both detecting and mediating the [Ca.sup.2+]/GTP signal for exocytotic membrane fusion.

The Alzheimer disease 40-residue amyloid [beta] protein (A[beta]P[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range of conductances ranging from 40 to 4000 pS. [Zn.sup.2+] has been reported to bind to A[beta]P[1-40] with high affinity, and it has been implicated in the formation of an amyloid plaques. We now report the functional consequences of such [Zn.sup.2+] binding for the A[beta]P[1-40] channel. Provided the A[beta]P[1-40] channel is expressed in the low conductance (400 pS), [Zn.sup.2+] doses in the millimolar range were required to exert substantial blockade. The [Zn.sup.2+] chelator o-phenanthroline reverses the blockade. We also found that [Zn.sup.2+] modulates A[beta]P[1-40] channel gating and conductance only from one side of the channel. These data are consistent with predictions of our recent molecular modeling studies on A[beta]P[1-40] channels indicating asymmetric [Zn.sup.2+]A[beta]P[1-40] interactions at the entrance to the pore.

Synthetic Alzheimer disease 40-residue amyloid beta-protien (A-beta-P), upon being introduced into planar lipid bilayers, undergoes molecular adaptation to form cation conductance channels. Fusion of liposomes comprising the peptides renders the activation of these channels. This amyloid ion-channel activity may be the underlying cause for cell lethality effecting amyloid neurotoxicity during Alzheimer's disease. The molecular pathway involved in A-beta-P channel induction may be ascribed to the generation of peptide complex.

Synthetic Alzheimerdisease amyloid beta-protein was incorporated in phosphatidylserine liposomes which were subsequently fused with a planar bilayer. Conductance analysis showed that the incorporated peptides formed cation-selective channels across the planar lipid bilayers. Determinations of permeability sequences of different cations showed that the amyloid beta-protein forms a multi-ion channel. It is proposed that this channel effect could explain the neurotoxic effects of amyloid beta-protein by disturbing ion, especially calcium ion, homeostasis.

The cystic fibrosistransmembrane regulator (CFTR) has been hypothesized to be the chloride channelwhich is defective in cystic fibrosis. To localize the anion channel activity within the protein, the first nucleotide binding fold (NBF-1) was expressed in bacteria, and the reconstituted recombinant protein was characterized. The results showed that the recombinant protein could bind to ATP, and expressed ATP-limited channel activity. These indicate that NBF-1 represents all or part of the anion channel activity of CFTR, which is modulated by ATP.

The ANX7 gene is located on human chromosome 10q21, a site long hypothesized to harbor a tumor suppressor gene(s) (TSG) associated with prostate and other cancers. To test whether ANX7 might be a candidate TSG, we examined the ANX7-dependent suppression of human tumor cell growth, stage-specific ANX7 expression in 301 prostate specimens on a prostate tissue microarray, and loss of heterozygosity (LOH) of microsatellite markers at or near the ANX7 locus. Here we report that human tumor cell proliferation and colony formation are markedly reduced when the wild-type ANX7 gene is transfected into two prostate tumor cell lines, LNCaP and DU145. Consistently, analysis of ANX7 protein expression in human prostate tumor microarrays reveals a significantly higher rate of loss of ANX7 expression in metastatic and local recurrences of hormone refractory prostate cancer as compared with primary tumors (P = 0.0001). Using four microsatellite markers at or near the ANX7 locus, and laser capture microdissected tumor cells, 35% of the 20 primary prostate tumors show LOH. The microsatellite marker closest to the ANX7 locus showed the highest rate of LOH, including one homozygous deletion. We conclude that the ANX7 gene exhibits many biological and genetic properties expected of a TSG and may play a role in prostate cancer progression. cancer genetics | chromosome 10q21 | loss of heterozygosity

The mammalian anx7 gene codes for a [Ca.sup.2+]-activated GTPase, which supports [Ca.sup.2+]/GTP-dependent secretion events and [Ca.sup.2+] channel activities in vitro and in vivo. To test whether anx7 might be involved in [Ca.sup.2+] signaling in secreting pancreatic [Beta] cells, we knocked out the anx7 gene in the mouse and tested the insulin-secretory properties of the [Beta] cells. The nullizygous anx7 (-/-) phenotype is lethal at embryonic day 10 because of cerebral hemorrhage. However, the heterozygous anx7 (+/-) mouse, although expressing only low levels of ANX7 protein, is viable and fertile. The anx7 (+/-) phenotype is associated with a substantial defect in insulin secretion, although the insulin content of the islets, is 8- to 10-fold higher in the mutants than in the normal littermate control. We infer from electrophysiological studies that both glucose-stimulated secretion and voltage-dependent [Ca.sup.2+] channel functions are normal. However, electrooptical recordings indicate that the (+/-) mutation has caused a change in the ability of inositol 1,4,5-trisphosphate ([IP.sub.3])-generating agonists to release intracellular calcium. The principle molecular consequence of lower anx7 expression is a profound reduction in [IP.sub.3] receptor expression and function in pancreatic islets. The profound increase in islets, [Beta] cell number, and size may be a means of compensating for less efficient insulin secretion by individual defective pancreatic [Beta] cells. This is a direct demonstration of a connection between glucose-activated insulin secretion and [Ca.sup.2+] signaling through [IP.sub.3]-sensitive [Ca.sup.2+] stores.

Digitoxin and structurally related cardiac glycoside drugs potently block activation of the TNF-α/NF-κB signaling pathway. We have hypothesized that the mechanism might be discovered by searching systematically for selective inhibitory action through the entire pathway. We report that the common action of these drugs is to block the TNF-α-dependent binding of TNF receptor 1 to TNF receptor-associated death domain. This drug action can be observed with native cells, such as HeLa, and reconstituted systems prepared in HEK293 cells. All other antiinflammatory effects of digitoxin on NF-κB and c-Jun N-terminal kinase pathways appear to follow from the blockade of this initial upstream signaling event. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is a fatal, autosomal, recessive genetic disease that is characterized by profound lung inflammation. The inflammatory process is believed to be caused by massive overproduction of the proinflammatory protein 11-8, and the high levels of 11-8 in the CF lung are therefore believed to be the central mechanism behind CF lung pathophysiology. We show here that digitoxin, at sub nM concentrations, can suppress hypersecretion of 11-8 from cultured CF lung epithelial cells. Certain other cardiac glycosides are also active but with much less potency. The specific mechanism of digitoxin action is to block phosphorylation of the inhibitor of NF-κB (lκBa). lκBα phosphorylation is a required step in the activation of the NF-κB signaling pathway and the subsequent expression of 11-8. Digitoxin also has effects on global gene expression in CF cells. Of the informative genes expressed by the CF epithelial cell line IB-3, 58 are significantly (P < 0.05) affected by gene therapy with wild-type (CFTR CF transmembrane conductance regulator). Of these 58 genes, 36 (62%) are similarly affected by digitoxin and related active analogues. We interpret this result to suggest that digitoxin can also partially mimic the genomic consequences of gene therapy with CF transmembrane conductance regulator. We therefore suggest that digitoxin, with its lengthy history of human use, deserves consideration as a candidate drug for suppressing 11-8-dependent lung inflammation in CF. [ABSTRACT FROM AUTHOR]