Epigenetic silencing of beta-spectrin, a TGF-beta signaling/scaffolding protein in a human cancer stem cell disorder: Beckwith-Wiedemann syndrome.

Hereditary cancer syndromes provide powerful insights into dysfunctional signaling pathways that lead to sporadic cancers. Beckwith-Wiedemann syndrome (BWS) is a hereditary human cancer stem cell syndrome currently linked to deregulated imprinting at chromosome 11p15 and uniparental disomy. However, causal molecular defects and genetic models have remained elusive to date in the majority of cases. The non-pleckstrin homology domain ß-spectrin (ß2SP) (the official name for human is Spectrin, beta, nonerythrocytic 1 (SPTBN1), isoform 2; the official name for mouse is Spectrin beta 2 (Spnb2), isoform 2), a scaffolding protein, functions as a potent TGF-ß signaling member adaptor in tumor suppression and development. Yet, the role of the ß2SP in human tumor syndromes remains unclear. Here, we report that ß2SP(+/-) mice are born with many phenotypic characteristics observed in BWS patients, suggesting that ß2SP mutant mice phenocopy BWS, and ß2SP loss could be one of the mechanisms associated with BWS. Our results also suggest that epigenetic silencing of ß2SP is a new potential causal factor in human BWS patients. Furthermore, ß2SP(+/-) mice provide an important animal model for BWS, as well as sporadic cancers associated with it, including lethal gastrointestinal and pancreatic cancer. Thus, these studies could lead to further insight into defects generated by dysfunctional stem cells and identification of new treatment strategies and functional markers for the early detection of these lethal cancers that otherwise cannot be detected at an early stage.

Motor Unit Number Estimate and Isometric Hand Grip Strength in Military Veterans with or Without Muscular Complaints: Reference Values for Longitudinal Follow-up.

INTRODUCTION: It remains unclear if Gulf War (GW) veterans have a higher risk of developing motor neuron disorder. We intended to establish baseline neurophysiological values, including thenar motor unit number estimate (MUNE) and isometric hand grip (IHG) strength, to compare future follow-ups of deployed GW veterans with or without muscular complaints. MATERIALS AND METHODS: We evaluated 19 GW veterans with self-reported weakness, cramps, or excessive muscle fatigue (Ill-19) and compared them with 18 controls without such muscular complaints (C-18). We performed MUNE on hand thenar muscles using adapted multipoint stimulation method for Ill-19 and 15 controls (C-15). We measured IHG strength (maximum force, endurance, and fatigue level) on Ill-19 and C-18 with a hand dynamometer. We performed nerve conduction studies on all study participants to determine which subjects had mild carpal tunnel syndrome (CTS). We compared the MUNE and IHG strength measures between Ill group and controls and between those with CTS and those without CTS. RESULTS: We obtained thenar MUNE of Ill-19 (95% CI of mean: 143-215; mean age: 46 yr) and compared it with that of C-15 (95% CI of mean: 161-230; mean age: 45 yr), and 95% of CI of mean among IHG strength variables (maximum force: 324-381 Newton; endurance: 32-42 s; fatigue level: 24%-33%) compared with C-18 (maximum force: 349-408 Newton; endurance: 35-46 s; fatigue level: 21%-27%). There was no significant difference in either MUNE or IHG strength between Ill-19 group and controls. The MUNE and IHG maximum forces were significantly lower in those with CTS compared with those without CTS. As a surrogate of mild CTS, the median versus ulnar distal sensory latency on nerve conduction study was only weakly associated with MUNE, maximum force, and fatigue level, respectively. CONCLUSION: To our knowledge, no published study on MUNE reference values of military veteran population has been available. The quantifiable values of both thenar MUNE and IHG strength of military veterans serve as baselines for our longitudinal follow-up of motor neuron function of deployed troops. These reference values are also useful for other laboratories to study veterans' motor system with or without mild CTS.

The benzamide derivative N-[1-(7-tert-Butyl-1H-indol-3-ylmethyl)-2-(4-cyclopropanecarbonyl-3-methyl-piperazin-1-yl)-2-oxo-ethyl]-4-nitro-benzamide (SP-10) reduces HIV-1 infectivity in vitro by modifying actin dynamics.

Current treatments for patients infected with HIV are suboptimal. There is a need for new HIV therapies that act through different mechanisms than current treatments. We investigated the in vitro efficacy, safety and mechanism of action of the benzamide derivative N-[1-(7-tert-Butyl-1H-indol-3-ylmethyl)-2-(4-cyclopropanecarbonyl-3-methyl-piperazin-1-yl)-2-oxo-ethyl]-4-nitro-benzamide (SP-10), a potential new HIV treatment. When HIV-1-responsive engineered HeLa cells were pre-incubated for 48 h with either SP-10 or zidovudine (AZT), SP-10 was able to inhibit viral replication at much lower concentrations (IC50 = 0.036 nM) than AZT (IC50 = 27.4 nM). In contrast to AZT, SP-10 also inhibited replication of the multidrug-resistant HIV-1 strain MDR-769 in the HeLa cell model. In co-incubation experiments, SP-10 also inhibited the CCR5-sensitive HIV-1 BaL virus replication in human peripheral blood mononuclear cells. SP-10 displayed very low toxicity compared with current antiviral treatments. Confocal laser scanning microscopy and immunoprecipitation studies showed that SP-10 reduced the expression of CD4 and CCR5 on the surface of the host cell. SP-10 also reduced the level of gp120 binding to the cell surface. Confocal laser scanning microscopy studies showed that SP-10 blocked the formation of actin filaments (F-actin) and altered actin accumulation near the cell surface. These promising results suggest that SP-10 has a novel mechanism of action that enables effective inhibition of HIV-1 binding and cell entry. Further development of SP-10 as a new HIV treatment appears warranted.

The spirostenol (22R, 25R)-20alpha-spirost-5-en-3beta-yl hexanoate blocks mitochondrial uptake of Abeta in neuronal cells and prevents Abeta-induced impairment of mitochondrial function.

Abeta(1-42) has been shown to uncouple the mitochondrial respiratory chain and promote the opening of the membrane permeability transition (MPT) pore, leading to cell death. We have previously reported that the spirostenol derivative (22R, 25R)-20alpha-spirost-5-en-3beta-yl hexanoate (SP-233) protects neuronal cells against Abeta(1-42) toxicity by binding to and inactivating the peptide. Picomolar concentrations of Abeta(1-42) decreased the mitochondrial respiratory coefficient in mitochondria isolated from the rat forebrain, and this decrease was partially reversed by SP-233. SP-233 abolished the uncoupling of oxidative phosphorylation induced by carbonyl cyanide 3-chlorophenylhydrazone on isolated mitochondria. These results are consistent with a direct effect of SP-233 on the MPT. Moreover, SP-233 displayed a neuroprotective effect on SK-N-AS human neuroblastoma cells treated with the MPT promoter, phenylarsine oxide. Treatment of SK-N-AS cells with Abeta(1-42) resulted in an accumulation of the peptide in the mitochondrial matrix; SP-233 completely scavenged Abeta(1-42) from the matrix. In addition, SP-233 protected the cells against mitochondrial toxins targeting complexes IV and V of the respiratory chain. These results indicate that Abeta(1-42) and SP-233 exert direct effects on mitochondrial function and SP-233 protects neuronal cells against Abeta-induced toxicity by targeting Abeta directly.

Identification, design, synthesis, and pharmacological activity of (4-ethyl-piperazin-1-yl)-phenylmethanone derivatives with neuroprotective properties against beta-amyloid-induced toxicity.

In search of novel therapeutic approaches for Alzheimer's disease (AD), we report herein the identification, design, synthesis, and pharmacological activity of (4-ethyl-piperaz-1-yl)-phenylmethanone derivatives with neuroprotective properties against beta-amyloid-induced toxicity. (4-ethyl-piperaz-1-yl)-phenylmethanone is a common substructure shared by molecules isolated from plants of the Asteraceae genus, traditionally used as restorative of lost or declining mental functions. (4-Ethyl-piperaz-1-yl)-phenylmethanone displayed strong neuroprotective properties against Abeta1-42 and reversed Abeta1-42-induced ATP depletion on neuronal cells, suggesting a mitochondrial site of action. Abeta1-42 has been described to induce a hyperactivity of the glutamate network in neuronal cells. (4-Ethyl-piperaz-1-yl)-phenylmethanone also inhibited the neurotoxic effect that glutamate displayed on PC12 cells, suggesting that the reduction of glutamate-induced neurotoxicity may be one of the mechanisms by which this compound exerts its neuroprotective properties against the deleterious effects of the Abeta1-42. These data suggest that the identified (4-ethyl-piperaz-1-yl)-phenylmethanone chemical entity exerts neuroprotective properties and may serve as a lead compound for the development of novel therapies for AD.

Identification of naturally occurring spirostenols preventing beta-amyloid-induced neurotoxicity.

22R-Hydroxycholesterol is an intermediate in the steroid biosynthesis pathway shown to exhibit a neuroprotective property against beta-amyloid (1-42) (Abeta) toxicity in rat PCl2 and human NT2N neuronal cells by binding and inactivating Abeta. In search of potent 22R-hydroxycholesterol derivatives, we assessed the ability of a series of naturally occurring entities containing the 22R-hydroxycholesterol structure to protect PC12 cells against Abeta-induced neurotoxicity, determined by measuring changes in membrane potential, mitochondrial diaphorase activity, ATP levels and trypan blue uptake. 22R-Hydroxycholesterol derivatives sharing a common spirost-5-en-3-ol or a furost-5-en-3-ol structure were tested. Although some of these compounds were neuroprotective against 0.1 microM Abeta, only three protected against the 1-10 microM Abeta-induced toxicity and, in contrast to 22R-hydroxycholesterol, all were devoid of steroidogenic activity. These entities shared a common structural feature, a long chain ester in position 3 and common stereochemistry. The neuroprotective property of these compounds was coupled to their ability to displace radiolabeled 22R-hydroxycholesterol from Abeta, suggesting that the Abeta-22R-hydroxycholesterol physicochemical interaction contributes to their beneficial effect. In addition, a 22R-hydroxycholesterol derivative inhibited the formation of neurotoxic amyloid-derived diffusible ligands. Computational docking simulations of 22R-hydroxycholesterol and its derivatives on Abeta identified two binding sites. Chemical entities, as 22R-hydroxycholesterol, seem to bind preferentially only to one site. In contrast, the presence of the ester chain seems to confer the ability to bind to both sites on Abeta, leading to neuroprotection against high concentrations of Abeta. In conclusion, these results suggest that spirost-5-en-3-ol naturally occurring derivatives of 22R-hydroxycholesterol might offer a new approach for Alzheimer's disease therapy.

Self-reported post-exertional fatigue in Gulf War veterans: roles of autonomic testing.

To determine if objective evidence of autonomic dysfunction exists from a group of Gulf War veterans with self-reported post-exertional fatigue, we evaluated 16 Gulf War ill veterans and 12 Gulf War controls. Participants of the ill group had self- reported, unexplained chronic post-exertional fatigue and the illness symptoms had persisted for years until the current clinical study. The controls had no self-reported post-exertional fatigue either at the time of initial survey nor at the time of the current study. We intended to identify clinical autonomic disorders using autonomic and neurophysiologic testing in the clinical context. We compared the autonomic measures between the 2 groups on cardiovascular function at both baseline and head-up tilt, and sudomotor function. We identified 1 participant with orthostatic hypotension, 1 posture orthostatic tachycardia syndrome, 2 distal small fiber neuropathy, and 1 length dependent distal neuropathy affecting both large and small fiber in the ill group; whereas none of above definable diagnoses was noted in the controls. The ill group had a significantly higher baseline heart rate compared to controls. Compound autonomic scoring scale showed a significant higher score (95% CI of mean: 1.72-2.67) among ill group compared to controls (0.58-1.59). We conclude that objective autonomic testing is necessary for the evaluation of self-reported, unexplained post-exertional fatigue among some Gulf War veterans with multi-symptom illnesses. Our observation that ill veterans with self-reported post-exertional fatigue had objective autonomic measures that were worse than controls warrants validation in a larger clinical series.

Dimethyl-carbamic acid 2,3-Bis-Dimethylcarbamoyloxy-6-(4-Ethyl-Piperazine-1-Carbonyl)-phenyl ester: a novel multi-target therapeutic approach to neuroprotection.

We report herein the synthesis and biological evaluation of dimethyl-carbamic acid 2,3-bis-dimethylcarbamoyloxy-6-(4-ethyl-piperazine-1-carbonyl)-phenyl ester (SP-04), a new drug candidate that is designed to offer a multi-target therapeutic neuroprotective approach as a treatment for Alzheimer's disease (AD). SP-04 inhibits acetylcholinesterase (AchE) activity both in vitro and in vivo, and induces a dose-dependent increase in Ach levels. SP-04 releases the metabolite 4-(4-ethyl-piperazin-1-yl)-1-(2,3,4-trihydroxy-phenyl)-butan-1-one (SP-04m). Both SP-04 and SP-04m are s1-receptor antagonists supporting their interest in relieving symptoms related to psychosis, a non-cognitive condition often associated with AD. SP-04m displays important antioxidant properties and both SP-04 and SP-04m offers neuroprotection against Ab42 toxicity in various neuronal cell lines. In addition, both SP-04 and SP-04m protect neuronal cells and rat brain mitochondria exposed to various mitochondrial respiratory chain complex toxins. Taken together these data suggest that the SP-04 multi-targeting approach might offer a novel therapeutic strategy for the treatment of AD.