Biosynthesis of enniatin B: partial purification and characterization of the synthesizing enzyme and studies of the biosynthesis.

Enniatin B-synthetase was purified 50-fold from Fusarium oxysporum strain ETH 1536/9. The biosynthesis of this depsipeptide seems to occur in a similar way to that of a number of peptide antibiotics like gramicidin S, tyrocidin and bacitracin. It has been shown that the single precursors of the molecule are activated in the form of thioesters via acyl adenylates. Further evidence will be presented, that N-methyl valine thioester bound to the enzyme is an obligatory intermediate in the biosynthetic process.     

Eco-friendly nonconjugated polymer dots for chemiluminometric determination of 4-nitrophenol

Polymer dots (PDs) are a new family of quantum dots for which their behavior and potential applications have not yet been completely explored. In this study, nonconjugated PDs were synthesized using a simple pyrolysis method and used for the chemiluminescence (CL) assay of 4-nitrophenol (4-NP). PDs increased the CL signal of the Ce(IV)–Na₂SO₃ reaction 39-fold. Using the CL spectrum, it was concluded that the emission at 434 nm was generated by excited PDs (PDs*), which are produced by energy transfer from SO₂* to PDs. Our experiments showed that 4-NP enhanced the CL signal of the Ce(IV)–Na₂SO₃–PDs reaction. The mechanism of this effect was explored by obtaining CL, ultraviolet–visible, and Fourier transform infrared spectra. Due to the high sensitivity and selectivity of the CL system for 4-NP, a probe was designed to determine 4-NP in the linear range 1.0–500 nmol/L with a detection limit of 0.33 nmol/L. Different spiked real samples were successfully analyzed using this probe.     

SIRT2 interferes with autophagy-mediated degradation of protein aggregates in neuronal cells under proteasome inhibition

Abnormal protein aggregates have been suggested as a common pathogenesis of many neurodegenerative diseases. Two well-known protein degradation pathways are responsible for protein homeostasis by balancing protein biosynthesis and degradative processes: the ubiquitin–proteasome system (UPS) and autophagy-lysosomal system. UPS serves as the primary route for degradation of short-lived proteins, but large-size protein aggregates cannot be degraded by UPS. Autophagy is a unique cellular process that facilitates degradation of bulky protein aggregates by lysosome. Recent studies have demonstrated that autophagy plays a crucial role in the pathogenesis of neurodegenerative diseases characterized by abnormal protein accumulation, suggesting that regulation of autophagy may be a valuable therapeutic strategy for the treatment of various neurodegenerative diseases. Sirtuin-2 (SIRT2) is a class III histone deacetylase that is expressed abundantly in aging brain tissue. Here, we report that SIRT2 increases protein accumulation in murine cholinergic SN56 cells and human neuroblastoma SH-SY5Y cells under proteasome inhibition. Overexpression of SIRT2 inhibits lysosome-mediated autophagic turnover by interfering with aggresome formation and also makes cells more vulnerable to accumulated protein-mediated cytotoxicity by MG132 and amyloid beta. Moreover, MG132-induced accumulation of ubiquitinated proteins and p62 as well as cytotoxicity are attenuated in siRNA-mediated SIRT2-silencing cells. Taken together, these results suggest that regulation of SIRT2 could be a good therapeutic target for a range of neurodegenerative diseases by regulating autophagic flux.     

Application of polyethyleneimine‐modified scaffolds to the regeneration of cartilaginous tissue

In this study, we analyzed the physicochemical and biophysical properties of three‐dimensional scaffolds modified using polyethyleneimine (PEI) and applied these scaffolds to the cultivation of bovine knee chondrocytes (BKCs). PEI was crosslinked in the bulk or on the surface of the ternary scaffolds comprising polyethylene oxide, chitin and chitosan. The results revealed that when the concentration of PEI was less than 300 μg/mL, the cytotoxicity of a scaffold was on the same order in the two method of modification. An increase in the concentration of PEI favored the adhesion of BKCs. When the amount of PEI in scaffolds is fixed, the surface‐modified scaffolds exhibited a higher adhesion efficiency of BKCs than the bulk‐modified scaffolds. For the regeneration of cartilaginous components, a higher amount of PEI in a scaffold yielded larger amounts of proliferated BKCs, secreted glycosaminoglycans, and produced collagen. In addition, the formation of neocartilage in the surface‐modified scaffolds was more effective than that in the bulk‐modified scaffolds. These tissue‐engineered scaffolds, modified by an appropriate concentration of PEI, can be potentially applied to cartilage repair in clinical trials. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Isolation of a 110 000 molecular weight protein in the purification of the nuclear chick intestinal 1,25-dihydroxyvitamin D-3 receptor

A single protein band of molecular weight 110 000 has been obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified 1,25-dihydroxyvitamin D-3 (1,25-(OH)₂D-3) receptor from crude nuclear extracts of chick intestinal mucosa, prepared in the presence of the protease inhibitors phenylmethylsulfonyl fluoride and ?-aminocaproic acid. The nuclear extract was subjected to a six-step purification scheme, involving polymin P and ammonium sulfate fractionation, DNA-cellulose affinity chromatography, Sephacryl S-200 gel filtration, blue dextran-Sepharose and a final DNA-cellulose chromatographic step. The receptor was obtained in about 1% yield and was purified approx. 3700-fold from the nuclear extract, as assessed by specific activity. Single peaks were observed with ³H-1,25-(OH)₂D-3-labeled crude nuclear extracts on Sephacryl S-200 gel filtration ($\text{Strokes′ radius}\text{= 35.5}\text{A}\text{?}$) and sucrose density gradient centrifugation (3.5 S). Although the identity of the M_r 110 000 protein will remain inconclusive until methods for further characterization are available, it may represent evidence for a higher molecular weight form of the 1,25-(OH)₂D-3 receptor than that observed previously.     

Cationic polyelectrolytes-lipases complexes formation as tool for recovery of these enzymes from their natural sources

In the present paper the formation of complexes between positively charge polyelectrolyte (polyethyleneimine and chitosan) and Candida rugosa lipase from a crude extract and porcine lipase from pancreas commercial homogenate preparations were analyzed. The solubility of lipases-cationic polyelectrolytes formation was dependent on: polyelectrolyte densities electrical charge, polyelectrolyte and enzyme concentrations and salts present in the solution. The lipase was recovered from the non-soluble complex by adding of NaCl at a given pH. Although the polyelectrolytes did not affect lipase biological activity, both of them produced good enzyme recovery (>90%); however, purification factors were low. This methodology appears to be a good previous prepurification and concentration method, using, low-cost polymers, allows the design of a purification method where the protein of interest is present in a large volume with respect to the small amount of polyelectrolyte added.     

Tau peptides and tau mutant protein aggregation inhibition by cationic polyethyleneimine and polyarginine

Tau protein plays a major role in Alzheimer's disease. The tau protein loses its functionality by self‐aggregation due to the two six‐amino acid sequences VQIVYK and VQIINK of the protein. Hence it is imperative to find therapeutics that could inhibit the self‐aggregation of this tau peptide fragments. Here, we study the inhibitory potential of a cationic polymer polyethyleneimine (PEI) and a cationic polypeptide arginine (Arg) on the aggregation of VQIVYK, and GKVQIINKLDL peptides, and tau mutant protein (P301L), found frequently in taupathy. Various characterization methods are employed including thioflavin S, transmission electron microscopy, and dynamic light scattering to study the aggregation/inhibition process in vitro. Results show that PEI and Arg significantly inhibit tau peptides and protein aggregation. The study could be applied to understand tau protein aggregation mechanism in the presence of cationic polymers.     

Polyethylenimine: A new differentiation factor to endothelial/cardiac tissue

Among different tissues, endothelial/cardiac types require specific factors to promote myocardial regeneration after occurred injuries. Herein, cardiac stem cells (CSCs) as the major cell population that involved in cardiovascular repair were selected to study the role of polyethyleneimine (PEI) agent on endothelial differentiation. After preparation of electrospun network of PEI with polyacrylonitrile, the related characterizations were carried out including scanning electron microscope (SEM), field-emission SEM, water contact angle, Fourier transform infrared spectroscopy and mechanical properties. Also, the release kinetic of the corresponding agent was studied up to 7 days. The cell differentiation studies were done in the following with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, Real-time polymerase chain reaction and immunostaining method. The whole obtained results approved the higher differentiation of CSCs into endothelial/cardiac cells. Finally, it is recommended that the PEI delivering increases the healing potency of CSCs and accordingly the regeneration speed of damaged cardiovascular tissue would be improved.     

Stimulation of initiation factor eIF-2 by a rat liver protein with GDPase activity

Phosphocellulose chromatography of initiation factor eIF-2 from rat liver separates it from a protein fraction which is highly stimulatory for [eIF-2.GTP.Met-tRNA_f] ternary complex formation. Evidence is presented which indicates that this stimulatory fraction contains a specific GDPase activity. eIF-2 dependent formation of 40S ribosomal initiation complexes is also enhanced by the GDPase preparation. The enzyme may play a role in the recycling of eIF-2 by removing inhibitory GDP which is generated during 80S initiation complex formation.