Small molecules differentiate definitive endoderm from human induced pluripotent stem cells on PCL scaffold.

Human induced pluripotent stem cells (hiPSCs) are attractive sources of cells for disease modeling in vitro, and they may eventually provide access to cells/tissues for the treatment of many degenerative diseases. Stepwise differentiation from hiPSCs to definitive endoderm (DE) will identify a key step in hepatocytes and beta cell development and may prove useful for transplantation therapy for liver diseases and diabetes. Inducer of definitive endoderm 1 (IDE1) is known to play an important role in the regional specification of DE. Here, we have investigated the effect of stimulation with IDE1 on the development of hiPSCs into DE cells in three-dimensional (3D) cultures. The differentiation was determined by immunofluorescence staining with Sox17, FoxA2, and goosecoid (Gsc) and also by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis. In this study, we showed that hiPSCs with 6-day IDE1 treatment (as chemical tool) on poly(ε-caprolactone) (PCL) nanofibrous scaffold were able to differentiate into DE cells.

The role of anionic peptide fragments in 1N4R human tau protein aggregation.

Cellular protein degradation systems are necessary to avoid the accumulation of misfolded or damaged proteins. Deficiency in these systems might cause to partial degradation of misfolded proteins and generation of amyloidogenic fragments. Protein misfolding is believed to be the primary cause of neurodegenerative disorders such as Alzheimer's disease (AD). In this study, we investigate effect of two anionic peptide fragments including, an acidic fragment of human Aß (Aß1-11) and a phosphorylated fragment of ß-Casein (Tetraphosphopeptide), on tau protein aggregation. According to our results, these peptide fragments, induced tau fibrillization in vitro. In sum, we suggest that structural and conformational characters of inducer are as important as charge distribution on anionic inducer molecules however more experiments would be need to exactly confirm this suggestion.

The effect of the crocus sativus L. Carotenoid, crocin, on the polymerization of microtubules, in vitro.

OBJECTIVE(S): Crocin, as the main carotenoid of saffron, has shown anti-tumor activity both in vitro and in vivo. Crocin might interact with cellular proteins and modulate their functions, but the exact target of this carotenoid and the other compounds of the saffron have not been discovered yet. Microtubular proteins, as one of the most important proteins inside the cells, have several functions in nearly all kinds of cellular processes. The aim of this study was to investigate whether crocin affects microtubule polymerization and tubulin structure. MATERIALS AND METHODS: Microtubules were extracted from sheep brains after two cycles of temperature-dependant assembly-disassembly in the polymerization buffer (PMG). Then phosphocellulose P11 column was used to prepare MAP-free tubulin. Turbidimetric assay of microtubules was performed by incubation of tubulins at 37 ºC in PIPES buffer. To investigate the intrinsic fluorescence spectra of tubulins, the emission spectra of tryptophans was monitored. To test the interaction of crocin with tubulin in more details, ANS has been used. RESULTS: Crocin extremely affected the tubulin polymerization and structure. Ultraviolet spectroscopy indicated that crocin increased polymerization of microtubules by nearly a factor of two. Fluorescence spectroscopic data also pointed to significant conformational changes of tubulin. CONCLUSION: We showed that crocin increased tubulin polymerization and microtubule nucleation rate and this effect was concentration dependant. After entering cell, crocin can modulate cellular proteins and their functions. Concerning the results of this study, crocin would be able to affect several cell processes through interaction with tubulin proteins or microtubules.

An in vitro study of the role of ß-boswellic acid in the microtubule assembly dynamics.

Structural integrity of microtubule protein (MTP) is pivotal for its physiological roles. Disruption of the MTP network is known to be involved in neurodegenerative disorders. The gum resin of plants of the boswellia species, with ß-boswellic acid (BBA) as the major component, has long been used in Ayurveda and Oriental Medicine to prevent amnesia. In the current study, we addressed the question whether BBA affects assembly dynamics behavior of tubulin. Our in vitro results revealed that BBA increases MTP length distribution and the polymerization rate of tubulin, moderately stabilizing it and diminishing both the critical concentration (C(c)) and the fraction of inactive tubulin (F(i)).

Altered tubulin assembly dynamics with N-homocysteinylated human 4R/1N tau in vitro.

Tau isoforms promote neuronal integrity through binding and stabilization of microtubule proteins (MTP). It has been shown that hyperphosphorylation of tau contributes to Alzheimer's disease (AD) pathology and related tauopathies. However, other pathogenic modifications of tau have not been well characterized. It is well accepted that elevated level of homocysteine (Hcy) is associated with neurodegenerative diseases such as AD. As a result of N-homocysteinylation of lysine residues, Hcy becomes a component of proteins, as a protein-homocystamide adduct, which affects protein structure and function. Here we demonstrate that N-homocysteinylation of human tau (4R/1N isoform) inhibits its function via impaired tau-tubulin specific binding and MTP assembly dynamics in vitro.

Effect of N-homocysteinylation on physicochemical and cytotoxic properties of amyloid ß-peptide.

Abstract Hyperhomocysteinemia has recently been identified as an important risk factor for Alzheimer's disease (AD). One of the potential mechanisms underlying harmful effects of homocysteine (Hcy) is site-specific acylation of proteins at lysine residues by homocysteine thiolactone (HCTL). The accumulation of amyloid ß-peptide (Aß) in the brain is a neuropathological hallmark of AD. In the present study we were interested to investigate the effects of N-homocysteinylation on the aggregation propensity and neurotoxicity of Aß(1-42). By coupling several techniques, we demonstrated that the homocysteinylation of lysine residues increase the neurotoxicity of the Aß peptide by stabilizing soluble oligomeric intermediates.

The enhancement effect of beta-boswellic acid on hippocampal neurites outgrowth and branching (an in vitro study).

Increasing evidences implicate impairment of axonal integrity in mechanisms underlying neurodegenerative disorders. Beta-boswellic acid (BBA) is the major component of Boswellia serrata gum. This resin has long been used in Ayurveda (India's traditional medicine) to prevent amnesia. In this study, the effect of BBA was examined on neurites outgrowth and branching as well as on polymerization dynamics of tubulin. The morphometric parameters (axonal length and neuritis branching) were examined microscopically after treating the hippocampal cells with BBA. Also the assembly process of tubulin was assessed using UV/V is spectrophotometer through following of absorbance at 350 nm. The results revealed that BBA could significantly enhance neurite outgrowth, branching, and tubulin polymerization dynamics. The obtained results suggest that enhancing effect of BBA on microtubule polymerization kinetics might be the origin of increasing axonal outgrowth and branching.