Cationic poly(amidoamine) promotes cytosolic delivery of bovine RNase A in melanoma cells, while maintaining its cellular toxicity

Ribonucleases are known to cleave ribonucleic acids, inducing cell death. RNase A, a member of the ribonuclease family, generally displayed poor in vitro activity. This has been attributed to factors such as low intracellular delivery. Poly(amidoamine)s have been used to promote the translocation of non-permeant proteins to the cytosol. Our objective was to demonstrate that poly(amidoamine)s could potentially promote the delivery of RNase A to selected cell line. Interactions of three cationic poly(amidoamine)s (P1, P2 and ISA1) with wild-type bovine RNase A were investigated using gel retardation assays, DLS and microcalorimetry. Although the polymers and the protein are essentially cationic at physiological pH, complexation between the PAAs and RNase A was observed. The high sensitivity differential scanning calorimetry (HSDSC) thermograms demonstrated that the thermal stability of the protein was reduced when complexed with ISA1 (Tmax decreased by 6.5 °C) but was not affected by P1 and P2. All the polymers displayed low cytotoxicity towards non-cancerous cells (IC50 > 3.5 mg mL?1). While RNase A alone was not toxic to mouse melanoma cells (B16F1), P1 was able to promote cytosolic delivery of biologically active RNase A, increasing cell death (IC50 = 0.09 mg mL?1)

Broadband Dielectric Spectroscopy on Human Blood

Dielectric spectra of human blood reveal a rich variety of dynamic processes. Achieving a better characterization and understanding of these processes not only is of academic interest but also of high relevance for medical applications as, e.g., the determination of absorption rates of electromagnetic radiation by the human body. The dielectric properties of human blood are studied using broadband dielectric spectroscopy, systematically investigating the dependence on temperature and hematocrit value. By covering a frequency range from 1 Hz to 40 GHz, information on all the typical dispersion regions of biological matter is obtained. We find no evidence for a low-frequency relaxation (alpha-relaxation) caused, e.g., by counterion diffusion effects as reported for some types of biological matter. The analysis of a strong Maxwell-Wagner relaxation arising from the polarization of the cell membranes in the 1-100 MHz region (beta-relaxation) allows for the test of model predictions and the determination of various intrinsic cell properties. In the microwave region beyond 1 GHz, the reorientational motion of water molecules in the blood plasma leads to another relaxation feature (gamma-relaxation). Between beta- and gamma-relaxation, significant dispersion is observed, which, however, can be explained by a superposition of these relaxation processes and is not due to an additional delta-relaxation often found in biological matter. Our measurements provide dielectric data on human blood of so far unsurpassed precision for a broad parameter range. All data are provided in electronic form to serve as basis for the calculation of the absorption rate of electromagnetic radiation and other medical purposes. Moreover, by investigating an exceptionally broad frequency range, valuable new information on the dynamic processes in blood is obtained.Comment: 17 pages, 9 figure

Dielectric relaxation spectroscopy of proteins in solution

Diese Arbeit beschreibt eine Untersuchung von Protein-Wasser Wechselwirkungen, Proteinfaltung und Proteinaggregation von gelösten Proteinen mittels dielektrischer Relaxationsspektroskopie (DRS). Anhand von DRS (MHz/GHz Bereich) der Modellsysteme Ribonuklease A und des amphiphilen Peptids N-acetyl-leucinamid wurden dabei neue Einblicke in die Dynamik der Protein-Wasser Wechselwirkung gewonnen. Untersuchungen der Proteinstabilität anhand des RNase-Harnstoff-Systems hat ergeben, dass die DRS sehr sensitiv auf die Protein-Harnstoffwechselwirkung reagiert. Wie die Ergebnisse der Aggregationsstudien von bovinem Insulin bei verschiedenen pH-Bedingungen zeigen, ist die DRS eine geeignete Methode um die Oligomerisation von Proteinenvon langreichweitigen elektrostatischer Wechselwirkungen zu unterscheiden.This thesis provides new dielectric relaxation spectroscopy (DRS) insights into protein-water interactions, protein folding and self-association of proteins in solutions. By carrying out comprehensive dielectric relaxation experiments in the MHz/GHz regime on a model protein; Ribonuclease A, and also on a model amphiphilic peptide; N-acety-leucine amide (NALA), it is shown that the juxtaposition of the current results with the other experimental and theoretical studies provide rational and unified view into protein-water dynamics. Further, to assess the protein stability we investigate RNase-Urea interactions and thereby demonstrate utility of DRS in unraveling subtle mechanisms action of urea on proteins. Finally, by considering the self-association of bovine insulin at acidic and basic pH we show that dielectric spectroscopy is a powerful experimental tool which can distinguish long-range interactions from protein oligomerisatio

Delignification of miscanthus using ethylenediamine (EDA) with or without ammonia and subsequent enzymatic hydrolysis to sugars.

Pretreatment of miscanthus is essential for efficient enzymatic production of cellulosic ethanol. This study reports a possible pretreatment method for miscanthus using aqueous ethylenediamine (EDA) for 30 min at 180 °C with or without ammonia. The mass ratio of miscanthus to EDA was varied from 1:3, 1:1, and 1:0.5, keeping the mass ratio of miscanthus to liquid (EDA + Water) constant at 1:8. The ammonia-to-miscanthus ratio was 1:0.25. After pretreatment with a ratio of 1:3 miscanthus to EDA, about 75 % of the lignin was removed from the raw miscanthus with 90 % retention of cellulose and 50 % of hemicellulose in the recovered solid. Enzymatic hydrolysis of the recovered solid miscanthus gave 63 % glucose and 62 % xylose conversion after 72 h. EDA provides an effective pretreatment for miscanthus, achieving good delignification and enhanced sugar yield by enzyme hydrolysis. Results using aqueous EDA with or without ammonia are much better than those using hot water and compare favorably with those using aqueous ammonia. The delignification efficiency of EDA pretreatment is high compared to that for hot-water pretreatment and is nearly as efficient as that obtained for aqueous-ammonia pretreatment

The Dielectric Response of Room-Temperature Ionic Liquids: Effect of Cation Variation

In continuation of recent work on the dielec. response of imidazolium-based ionic liqs. (ILs) (J. Phys. Chem. B, 2006, 110, 12682), the authors report on the effect of cation variation on the frequency-dependent dielec. permittivity up to 20 GHz of ionic liqs. The salts are comprised of pyrrolidinium, pyridinium, tetraalkylammonium, and triethylsulfonium cations combined with the bis((trifluoromethyl)sulfonyl)imide anion. The dielec. spectra resemble those obsd. for imidazolium salts with the same anion. In all cases, the major contribution results from a diffusive low-frequency response on the time scale of several 100 ps, which shows a broadly distributed kinetics similar to that of spatially heterogeneous states in supercooled and glassy systems rather than that obsd. in fluid systems. There is evidence for a weak secondary process near 10-20 ps. Perhaps the most interesting difference to imidazolium salts is founded in the missing portions of the spectra due to processes beyond the upper cutoff frequency of 20 GHz. These are lower than that obsd. for imidazolium-based salts and seem to vanish for tetraalkylammonium and triethylsulfonium salts. As for imidazolium salts, the extrapolated static dielec. consts. are on the order of eS equiv. 10-13, Classifying these ILs as solvents of moderate polarity

Effect of Mixed Acid Catalysis on Pretreatment and Enzymatic Digestibility of Sugar Cane Bagasse

Aqueous pretreatment using homogeneous acid catalyst is considered as a low-cost technology in the production of lignocellulosic bioethanol. To establish the synergism of mixed acids, pilot-level aqueous pretreatments of bagasse covering a wide range of combined severity (CS) were carried out. To investigate the effect of application of mixture of acids on xylose hydrolysis as well as glucose hydrolysis via pretreatment and enzymatic hydrolysis, the following three combinations of acids were explored: (1) oxalic acid + sulfuric acid (organic + mineral acid), (2) phosphoric + sulfuric acid (mineral acids), and (3) ferric chloride + sulfuric acid (Lewis acid with a mineral acid). Of the pretreatments evaluated, the synergism was most pronounced for the combination of sulfuric and phosphoric acid, which resulted in more than 90% conversion of hemicellulose to xylose and 70% conversion of cellulose to glucose through enzymatic hydrolysis. Fourier transform infrared (FTIR) studies of pretreated samples showed higher syringyl/guaiacyl (S/G) ratio for sulfuric and phosphoric acid combination pretreatment, leading to higher enzymatic conversion. FTIR and dynamic light scattering (DLS) experiments conducted on pretreated sugar cane bagasse provided useful correlation with regard to the pretreatment type, particle size, and enzymatic hydrolysis

Chemocatalytic Upgrading of Tailored Fermentation Products Toward Biodiesel

Biological and chemocatalytic processes are tailored in order to maximize the production of sustainable biodiesel from lignocellulosic sugar. Thus, the combination of hydrotalcite-supported copper(II) and palladium(0) catalysts with a modification of the fermentation from acetone-butanol-ethanol to isopropanol-butanol-ethanol predictably produces higher concentrations of diesel-range components in the alkylation reaction