Biophysical characteristics of neurotensin polyplex for in vitro and in vivo gene transfection

Previously we improved the neurotensin (NT)-polyplex by the coupling of HA2 fusogenic peptide (FP) and Vp1 SV40 karyophilic peptide (KP). We now report the proportion of [(125)I]-NT, [(3)H]-FP, and poly-l-lysine (PLL) in the NT-polyplex, and some of its biophysical properties. We concluded that the most efficient NT-polyplex comprised 1 NT, 4 FP, and 2 PLL molecules. Electrophoresis revealed that high acidity is detrimental for NT-polyplex stability. Electron microscopy and electrophoresis studies showed that 6 muM KP and 1% serum condensed the plasmid DNA (pDNA) before the appearance of toroid structures. Four plasmids were used to evaluate the transfection efficiency. In vitro, maximum expression was produced at molar ratios (pDNA : [(125)I]-NT-[(3)H]-FP-PLL conjugate) of 1:34 for pEGFP-N1 and 1:27 for pECFP-Nuc. Cotransfection of those plasmids was attained at their optimum molar ratios. In vivo, maximum expression of the pDAT-BDNF-flag in dopamine neurons was produced at a 1:45 molar ratio, whereas that of pDAT-EGFP was at 1:20. The NT-polyplex in the presence of 1 muM SR-48692, an NT-receptor specific antagonist, and untargeted polyplex did not cause transfection in vivo demonstrating the specificity of gene transfer via NT-receptor endocytosis. This information is essential for synthesizing an efficient NT-polyplex that can provide a useful tool for specific gene transfection.     

Solid-phase synthesis and structure-activity relationships of novel biarylethers as melanin-concentrating hormone receptor-1 antagonists

Melanin-concentrating hormone (MCH) is a cyclic 19 amino acid orexigenic neuropeptide. The action of MCH on feeding is thought to involve the activation of its respective G protein-coupled receptor MCH-R1. Consequently, antagonists that block MCH regulated MCH-R1 activity may provide a viable approach to the treatment of diet-induced obesity. This communication reports the discovery of a novel MCH-R1 receptor antagonist, the biarylether 7, identified through high throughput screening. The solid-phase synthesis and structure-activity relationship of related analogs is described.     

Frequent Seizures Are Associated with a Network of Gray Matter Atrophy in Temporal Lobe Epilepsy with or without Hippocampal Sclerosis

Objective

Patients with temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) have diffuse subtle gray matter (GM) atrophy detectable by MRI quantification analyses. However, it is not clear whether the etiology and seizure frequency are associated with this atrophy. We aimed to evaluate the occurrence of GM atrophy and the influence of seizure frequency in patients with TLE and either normal MRI (TLE-NL) or MRI signs of HS (TLE-HS).

Methods

We evaluated a group of 172 consecutive patients with unilateral TLE-HS or TLE-NL as defined by hippocampal volumetry and signal quantification (122 TLE-HS and 50 TLE-NL) plus a group of 82 healthy individuals. Voxel-based morphometry was performed with VBM8/SPM8 in 3T MRIs. Patients with up to three complex partial seizures and no generalized tonic-clonic seizures in the previous year were considered to have infrequent seizures. Those who did not fulfill these criteria were considered to have frequent seizures.

Results

Patients with TLE-HS had more pronounced GM atrophy, including the ipsilateral mesial temporal structures, temporal lobe, bilateral thalami and pre/post-central gyri. Patients with TLE-NL had more subtle GM atrophy, including the ipsilateral orbitofrontal cortex, bilateral thalami and pre/post-central gyri. Both TLE-HS and TLE-NL showed increased GM volume in the contralateral pons. TLE-HS patients with frequent seizures had more pronounced GM atrophy in extra-temporal regions than TLE-HS with infrequent seizures. Patients with TLE-NL and infrequent seizures had no detectable GM atrophy. In both TLE-HS and TLE-NL, the duration of epilepsy correlated with GM atrophy in extra-hippocampal regions.

Conclusion

Although a diffuse network GM atrophy occurs in both TLE-HS and TLE-NL, this is strikingly more evident in TLE-HS and in patients with frequent seizures. These findings suggest that neocortical atrophy in TLE is related to the ongoing seizures and epilepsy duration, while thalamic atrophy is more probably related to the original epileptogenic process.     

Biochemical effects of bleomycin A2 on Novikoff hepatoma ascites cells.

Purified nucleolar DNA was markedly degraded at a concentration of 13 mug/ml by bleomycin A2; bleomycin concentrations 20-30 times greater were required to degrade nucleoplasmic DNA. Whole nuclear DNA was degraded to only a small extent at 13 mug/ml but was markedly degraded at higher bleomycin concentrations. Treatment of the various types of DNA with high concentrations of bleomycin A2 produced low molecular weight (approximately 6S) fragments that were no longer sensitive to degradation by bleomycin A2. Hybridization studies demonstrated a loss of ribosomal DNA sequences from nucleolar DNA treated with bleomycin A2 in vitro. Studies on RNA synthesis in Novikoff hepatoma ascites cells in vitro showed there was a decreased uptake of 32Pi into high molecular weight nuclear RNA in the presence of bleomycin A2. These results indicate that nucleolar function is inhibited by a direct effect of bleomycin A2 on nucleolar DNA.     

Methyl Sulfone Induces Loss of Metastatic Properties and Reemergence of Normal Phenotypes in a Metastatic Cloudman S-91 (M3) Murine Melanoma Cell Line

Background

The most deadly form of cancer is not lung or colon, breast or prostate; it is any cancer that has become metastatic. Mortality due to metastatic melanoma, one of the most aggressive and deadly cancers, has increased steadily over the last several decades. Unfortunately, the arsenal of chemotherapeutic agents available today is most often unsuccessful at extending and improving the life expectancy of afflicted individuals. We sought to identify an effective and nontoxic agent against metastatic melanoma.

Methodology/Principal Findings

We chose to study Cloudman S-91 mouse melanoma cells (sub-clone M3, CCL53.1) because these cells are highly aggressive and metastatic, representing one of the deadliest types of cancer. Melanoma cells also had an experimental advantage because their morphology, which is easily monitored, relates to the physiology of metastatic cells and normal melanocytes. We chose to test methyl sulfone as a chemotherapeutic agent for two reasons. Because of its chemical structure, we speculated a potential anti-cancer activity by targeting microtubules. Equally important, methyl sulfone has a well-established safety profile in humans. Surprisingly, we found that malignant melanoma cells exposed to methyl sulfone demonstrated the loss of phenotypes characteristic of malignant cells, and the reemergence of phenotypes characteristic of healthy melanocytes. Briefly, over time methyl sulfone induced contact inhibition, loss of ability to migrate through an extracellular matrix, loss of anchorage-independent growth, proper wound healing followed by contact inhibition, irreversible senescence followed by arborization with melanosomes in arbors as seen in normal melanocytes.

Conclusions/Significance

Methyl sulfone may have clinical potential as a non-toxic agent effective against metastatic melanoma. Additionally, methyl sulfone has promise as a tool to explore molecular mechanisms of metastatic transformation as well as fundamental processes such as cell migration, contact inhibition, wound healing and cellular senescence.