Optimized liposomes with transactivator of transcription peptide and anti-apoptotic drugs to target hippocampal neurons and prevent tau-hyperphosphorylated neurodegeneration.

Liposomes (lip) carrying pharmaceuticals have shown promise in their ability to advance the therapy for neurodegenerative diseases. However, the low nerve-targeting capacity and poor penetration rate of lip through the blood-brain barrier (BBB) are major hurdles to achieving successful treatment. Herein, we developed lip incorporating cardiolipin (CL) and phosphatidic acid (PA) to promote their capability against hyperphosphorylation of tau protein, and a transactivator of transcription (TAT) peptide to permeate the BBB for delivering nerve growth factor (NGF), rosmarinic acid (RA), curcumin (CURC) and quercetin (QU). We derived an optimization method to assess a better composition of phospholipids in the lip loaded with the four medicines. Experimental results revealed that this optimized lip increased the viability of SK-N-MC cells insulted with ß-amyloid peptide (Aß) fibrils and prevented Wistar rat brain from producing hyperphosphorylated tau. CL and PA and the grafted TAT peptide on the carrier surface improved the rescue efficiency by inhibiting Aß deposition and reducing the expressions of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), c-Jun N-terminal protein kinase, p38, tau at serine 202 and caspase-3. The lip also enhanced the expressions of p-ERK5 and p-cyclic adenosine monophosphate response element-binding protein. The amalgamated activity of NGF, RA, CURC and QU, and the effect of charged CL/PA on Aß deposits supported the therapeutic efficacy of lip. The optimized TAT-NGF-RA-CURC-QU-CL/PA-lip can be a capable drug delivery system to cross the BBB and protect Alzheimer's disease brains from tau hyperphosphorylation. STATEMENTS OF SIGNIFICANCE: The therapeutic efficiency of liposomes (lip) against neurodegenerative disorder depends on their nerve-targeting capacity and ability to permeate the blood-brain barrier (BBB). Lip was developed incorporating cardiolipin (CL) and phosphatidic acid (PA) to promote their target specificity against hyperphosphorylation of tau protein, and a transactivator of transcription (TAT) peptide to permeate the BBB. We have successfully derived an optimization method using a new mathematical expression for the first time to assess a better composition of phospholipids in lip loaded with nerve growth factor (NGF), rosmarinic acid (RA), curcumin (CURC) and quercetin (QU). The optimized TAT-NGF-RA-CURC-QU-CL/PA-lip efficaciously down-regulated the expressions of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), c-Jun N-terminal protein kinase, p38, tau at serine 202 and caspase-3, and up-regulated the expressions of p-ERK5 and p-cyclic adenosine monophosphate response element-binding protein in Alzheimer's disease Wistar rat model.

Hepatitis B virus X protein and proinflammatory cytokines synergize to enhance TRAIL-induced apoptosis of renal tubular cells by upregulation of DR4.

Persistent infection with hepatitis B virus (HBV) may lead to HBV-associated glomerulonephritis (HBV-GN). Presence of HBV-DNA and -RNA in renal tubular epithelial cells (RTECs) suggests direct virus-induced injury. Increase in proinflammatory cytokines is also observed under these conditions. Apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plays a significant role in the pathogenesis of HBV-infections. However, the effects of HBV X protein (HBx) on TRAIL-induced apoptosis of RTECs especially under certain inflammatory conditions remain obscure. Here, we show that HBx synergizes with proinflammatory cytokines to significantly increase TRAIL-induced apoptosis of RTECs. HBx markedly up-regulates death receptor-4 (DR4) expression by enhancing the activation of nuclear factor-kappa B (NF-κB) in the presence of proinflammatory cytokines. Dramatic increase in DR4 expression leads to the sensitization of RTECs to TRAIL-induced apoptosis. Furthermore, in patients with HBV-GN, DR4 expression in the kidneys is significantly elevated and is positively correlated with the HBx and proinflammatory cytokines expression. These findings provide a novel insight into the underlying mechanisms of renal tubule lesions induced by HBx in HBV-GN.

[Preliminary study on transdermal characteristics and sunface anesthetic effects of lidocaine hydrochloride loaded trans-activator of transcription peptide conjugated nano-niosome in animals].

OBJECTIVE: To prepare a new dental topical anesthetics, lidocaine hydrochloride loaded trans-activator of transcription peptide conjugated nano-niosome (LID-TAT-N), and to evaluate its transdermal properties and topical anesthesia effects. METHODS: LID-TAT-N was prepared using reverse-phase evaporation method, and lidocaine loaded conventional liposome (LID-CL) was prepared in the same manner as positive control. The diameter, ζ potential and encapsulation efficiency of LID-TAT-N and LID-CL were measured. The skin permeation of LID-TAT-N was examined, and compared with LID-CL and lidocaine injection (LID-IJ, as negative control), using a Franz diffusion cell mounted with depilated mouse skin in vitro for 12 hours. Each experiment was repeated six times. The anesthetic effect of the new topical anesthetic was investigated on the cornea of rabbits. RESULTS: The mean diameter of LID-TAT-N was smaller than that of LID-CL [(152.7 ± 10.6) nm vs. (259.5 ± 15.5) nm, P < 0.01]. The 12 h cumulative permeation amount was significantly higher in LID-TAT-N group [(1 340.0 ± 97.5) µg · cm(-2)] than those of LID-CL and LID-IJ groups [(1 060.6 ± 80.2), (282.6 ± 65.1) µg · cm(-2), respectively, P < 0.05]. Rabbit corneal reflex results showed that LID-TAT-N had anesthetic effect and the duration of analgesia [(24.8 ± 2.8) min] was also longer than that of LID-IJ [(14.5 ± 2.3) min, P < 0.05]. CONCLUSIONS: LID-TAT-N had good transdermal ability, and the advanced skin penetration feature can improve its tropical anesthetic effect.

Probing the electrostatics and pharmacological modulation of sequence-specific binding by the DNA-binding domain of the ETS family transcription factor PU.1: a binding affinity and kinetics investigation.

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤10(5)M(-)(1)s(-)(1)), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>10(7)M(-)(1)s(-)(1)). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes.

Correlation of Wnt-2 expression and beta-catenin intracellular accumulation in Chinese gastric cancers: relevance with tumour dissemination.

Wnt/beta-catenin signalling pathway is integrally associated with human tumour development and progression. Aberrant beta-catenin intracellular distribution has been found in gastric cancer, but the pattern of Wnt expression in stepwise gastrocarcinogenesis and its potential influence in beta-catenin distribution are still lesser known. By the methods of frozen tissue array-based immunohistochemistry, Western blot analysis and RT-PCR, a paralleled study was conducted to check Wnt2 expression and beta-catenin intracellular distribution in two major subtypes of gastric cancers (intestinal gastric cancer, i-GC and diffuse gastric cancer, d-GC) and their premalignant (intestinal metaplasia, IM and chronic gastritis, CG) and noncancerous counterparts. According to the results obtained and the clinical data collected, correlation of Wnt2 expression with beta-catenin translocalisation and their links with tumour dissemination were elucidated. The results demonstrated (1) that Wnt2 expression and cytoplasmic/nuclear beta-catenin accumulations appeared in most gastric cancers irrespective to their morphological phenotypes, (2) that over-expressed Wnt and nuclear translocalisation of beta-catenin were found in 68 and 58% of i-GCs and in 47 and 47% of d-GCs in a closely related pattern (P<0.01) and (3) that co-existence of Wnt2 up-regulation/beta-catenin nuclear translocalisation were positively associated with lymph node metastasis (P<0.05) as well as T-stage. These data indicate that Wnt/beta-catenin signalling pathway is activated in most of gastric cancers, which may play pivotal roles either in gastric cancer formation or in tumour invasion and dissemination.

VEGF-induced paracellular permeability in cultured endothelial cells involves urokinase and its receptor.

Vascular endothelial growth factor/vascular permeability factor (VEGF) has been implicated in blood/tissue barrier dysfunctions associated with pathological angiogenesis, but the mechanisms of VEGF-induced permeability increase are poorly understood. Here, the role of VEGF-induced extracellular proteolytic activities on the endothelial cell permeability increase is evaluated. Confluent monolayers of bovine retinal microvascular endothelial (BRE) cells grown on porous membrane were treated with VEGF or urokinase plasminogen activator (uPA), and permeability changes were analyzed. uPA-induced permeability was rapid and sustained, but VEGF-induced permeability showed a biphasic pattern: a rapid and transient phase (1-2 h) followed by delayed and sustained phase (6-24 h). The delayed, but not the early phase of VEGF-induced permeability, was blocked by anti-uPA or anti-uPAR (uPA receptor) antibodies and was accompanied by reduced transendothelial electrical resistance, indicating the paracellular route of permeability. Confocal microscopy and Western blotting showed that VEGF treatment increased free cytosolic beta-catenin, which was followed by beta-catenin nuclear translocation, upregulation of uPAR, and downregulation of occludin. Membrane-bound occludin was released immediately after uPA treatment, but with a long delay after VEGF treatment, suggesting a requirement for uPAR gene expression. In conclusion, VEGF induces a sustained paracellular permeability in capillary endothelial cells that is mediated by activation of the uPA/uPAR system.

Long-acting forms of Sonic hedgehog with improved pharmacokinetic and pharmacodynamic properties are efficacious in a nerve injury model.

The therapeutic effects of the Sonic hedgehog (Shh) have been difficult to evaluate because of its relatively short serum half-life. To address this issue polyethylene glycol modification (PEGylation) was investigated as an approach to improve systemic exposure. Shh was PEGylated by a targeted approach using cysteines that were engineered into the protein by site-directed mutagenesis as the sites of attachment. Sixteen different versions of the protein containing one, two, three, or four sites of attachment were characterized. Two forms were selected for extensive testing in animals, Shh A192C, which provided a single site for PEGylation, and Shh A192C/N91C, which provided two sites. The PEGylated proteins were evaluated for reaction specificity by SDS-PAGE and peptide mapping, in vitro potency, pharmacokinetic and pharmacodynamic properties, and efficacy in a sciatic nerve injury model. Targeted PEGylation was highly selective for the engineered cysteines and had no deleterious effect on Shh function in vitro. Systemic clearance values in rats decreased from 117.4 mL/h/kg for unmodified Shh to 29.4 mL/h/kg for mono-PEGylated Shh A192C that was modified with 20 kDa PEG-maleimide and to 2.5 mL/h/kg for di-PEGylated Shh A192C/N91C modified with 2, 20 kDa PEG vinylsulfone adducts. Serum half-life increased from 1 h for unmodified Shh to 7.0 and 12.6 h for the mono- and di-PEGylated products. These changes in clearance and half-life resulted in higher serum levels of Shh in the PEG-Shh-treated animals. In Ptc-LacZ knock-in mice expressing lacZ under regulation of the Shh receptor Patched, about a 10-fold lower dose of PEG-Shh was needed to induce beta-galactosidase than for the unmodified protein. Therapeutic treatment of mice with PEG-Shh enhanced the regeneration of injured sciatic nerves. These studies demonstrate that targeted PEGylation greatly alters the pharmacokinetic and pharmacodynamic properties of Shh, resulting in a form with improved pharmaceutical properties.

Selective activation of an apoptotic retinoid precursor in macrophage cell lines.

Advances in the understanding of the retinoid signaling mechanism has allowed the discovery of highly selective retinoids that activate only one specific receptor class, subtype, or signaling pathway. These novel compounds lack certain of the common retinoid toxicities and therefore suggest promising new approaches for therapeutic applications. We describe here a new compound, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid methyl ester (MX84), that is selectively activated in macrophages, leading to killing of only macrophage monocyte type cells in vitro. We provide evidence that MX84 is an inactive precursor that is converted into an active apoptosis-inducing retinoid in macrophages. The macrophage activity is also secreted, and our data suggest that the secreted activity is a phospholipase D type activity. Our observation may lead to the development of molecules that are highly macrophage-selective apoptosis inducers in vivo and that could represent important novel therapeutics against diseases caused by excessive macrophage activity.

Functional immobilization of a DNA-binding protein at a membrane interface via histidine tag and synthetic chelator lipids.

The coupling of a DNA-binding protein to self-organized lipid monolayers is examined at the air-water interface by means of film balance techniques and epifluorescence microscopy. We used two recombinant species of the heat shock factor HSF24 which differ only in a carboxy-terminal histidine tag that interacts specifically with the nickel-chelating head group of a synthetic chelator lipid. As key function, HSF24 binds to DNA that contains heat-shock responsible promoter elements. In solution, DNA-protein complex formation is demonstrated for the wild type and fusion protein. Substantial questions of these studies are whether protein function is affected after adsorption to lipid layers and whether a specific docking via histidine tag to the chelator lipid leads to functional immobilization. Using lipid mixtures that allow a lateral organization of chelator lipids within the lipid film, specific binding and unspecific adsorption can be distinguished by pattern formation of DNA-protein complexes. At the lipid interface, functional DNA-protein complexes are only detected, when the histidine-tagged protein was immobilized specifically to a chelator lipid containing monolayer. These results demonstrate that the immobilization of histidine-tagged biomolecules to membranes via chelator lipids is a promising approach to achieve a highly defined deposition of these molecules at an interface maintaining their function.