Photoluminescent biocompatible silicon nanoparticles for cancer theranostic applications

Silicon nanoparticles (SiNPs) obtained by mechanical grinding of porous silicon have been used for visualization of living cells in vitro. It was found that SiNPs could penetrate into the cells without any cytotoxic effect up to the concentration of 100 μg/ml. The cell cytoplasm was observed to be filled by SiNPs, which exhibited bright photoluminescence at 1.6 eV. SiNPs could also act as photosensitizers of the singlet oxygen generation, which could be used in the photodynamic therapy of cancer. These properties of SiNPs are discussed in view of possible applications in theranostics (both in therapy and in diagnostics). (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Biodegradation of glyphosate by soil bacteria: Optimization of cultivation and the method for active biomass storage

Conditions for obtaining the active biomass of Ochrobactrum anthropi GPK 3 and Achromobacter sp. Kg 16, bacteria which are able to degrade the herbicide glyphosate (N-phosphonomethylglycine), were investigated. In the batch culture, degradation was most effective in the medium with pH 6.0–7.0 and aeration at 10–60% of air saturation supplemented with glutamate and ammonium chloride as sources of carbon and nitrogen, respectively. Due to the adaptation of the cells and induction of the relevant enzymatic systems, the inoculum grown in the presence of glyphosate exhibited 1.5–2-fold higher efficiency of xenobiotic degradation than that grown with other sources of phosphorus (orthophosphate and methylphosphonic acid). The efficiency of the toxicant decomposition increased with an increase in a specific load of glyphosate, which the cells were subjected to during the initial stage of growth. The specific load was regulated both by the initial cell concentration and the concentration of the phosphorus source, and the effect was probably determined by its availability to microorganisms. Storage of the liquid biopreparation as a paste with stabilizers (ascorbate, thiourea, and glutamate) at room temperature for 50 days resulted in high level of bacteria viability and a degrading activity approximately equal to that obtained when the bacteria were maintained on the agar medium containing glyphosate at 4°C with monthly transfers to the fresh culture medium.     

Characteristics of immunoaffinity chromatography and a new method for isolation of human thyroid peroxidase from subcellular fractions of thyroid gland

A system for quantitative determinations of human thyroid peroxidase (TPO) in biological fluids has been obtained, based on the use of enzyme-linked immunosorbent assay. The immunochemical properties of TPO were studied under variable conditions and a new method for isolating the protein from the microsomes, mitochondria, and cytosol of thyroid glands of patients with diverse thyroid diseases was developed. The procedure involves solubilization of subcellular fractions with detergents, their sonication, two sequential runs of chromatography (on sorbents with immobilized monoclonal antibodies against TPO and goat anti-human immunoglobulin antibodies), treatment with ribonuclease, and dialysis. Highly purified preparations of intact TPO and a product of its limited trypsinolysis are expected to be used as research tools and components of high-sensitivity immunoassays.     

Asymmetry in the lipid affinity of bihelical amphipathic peptides. A structural determinant for the specificity of ABCA1-dependent cholesterol efflux by peptides

ApoA-I contains a tandem array of amphipathic helices with varying lipid affinity, which are critical in its ability to bind and remove lipids from cells by the ABCA1 transporter. In this study, the effect of asymmetry in the lipid affinity of amphipathic helices in a bihelical apoA-I mimetic peptide, 37pA, on lipid efflux by the ABCA1 transporter was examined. Seven peptide variants of 37pA were produced by substituting a varying number of hydrophobic amino acids for alanine on either one or both helices. The 5A peptide with five alanine substitutions in the second helix had decreased helical content compared with 37pA (5A, 12+/-1% helicity; 37pA, 28+/-2% helicity) and showed less self-association but, similar to the parent peptide, was able to readily solubilize phospholipid vesicles. Furthermore, 5A, unlike the parent peptide 37pA, was not hemolytic (37pA, 27+/-2% RBC lysis, 2 h, 18 microm). Finally, the 5A peptide stimulated cholesterol and phospholipid efflux by the ABCA1 transporter with higher specificity (ABCA1-transfected versus untransfected cells) than 37pA (5A, 9.7+/-0.77%, 18 h, 18 microm versus 1.5+/-0.27%, 18 h, 18 microm (p<0.0001); 37pA, 7.4+/-0.85%, 18 h, 18 microm versus 5.8+/-0.20%, 18 h, 18 microm (p=0.03)). In summary, we describe a novel bihelical peptide with asymmetry in the lipid affinity of its helices and properties similar to apoA-I in terms of specificity for cholesterol efflux by the ABCA1 transporter and low cytotoxicity.     

A mouse model of harlequin ichthyosis delineates a key role for Abca12 in lipid homeostasis

Harlequin Ichthyosis (HI) is a severe and often lethal hyperkeratotic skin disease caused by mutations in the ABCA12 transport protein. In keratinocytes, ABCA12 is thought to regulate the transfer of lipids into small intracellular trafficking vesicles known as lamellar bodies. However, the nature and scope of this regulation remains unclear. As part of an original recessive mouse ENU mutagenesis screen, we have identified and characterised an animal model of HI and showed that it displays many of the hallmarks of the disease including hyperkeratosis, loss of barrier function, and defects in lipid homeostasis. We have used this model to follow disease progression in utero and present evidence that loss of Abca12 function leads to premature differentiation of basal keratinocytes. A comprehensive analysis of lipid levels in mutant epidermis demonstrated profound defects in lipid homeostasis, illustrating for the first time the extent to which Abca12 plays a pivotal role in maintaining lipid balance in the skin. To further investigate the scope of Abca12's activity, we have utilised cells from the mutant mouse to ascribe direct transport functions to the protein and, in doing so, we demonstrate activities independent of its role in lamellar body function. These cells have severely impaired lipid efflux leading to intracellular accumulation of neutral lipids. Furthermore, we identify Abca12 as a mediator of Abca1-regulated cellular cholesterol efflux, a finding that may have significant implications for other diseases of lipid metabolism and homeostasis, including atherosclerosis.     

Action of nonviral gene delivery vectors on human complement system: low anticomplementary activity of lipoplexes based on lacZ plasmid and phospholipid/oligocation liposomes

A simple test-system has been developed for the first time in order to detect the ability of effectors (lipoplexes) to activate the complement system in an antibody-independent manner to serve as acceptors of nascent C4b and to inhibit formation of the key enzyme of complement, C3-convertase. The effect of plasmid DNA (pCMV-SPORT-LacZ), negatively charged cardiolipin (CL), neutral phosphatidylcholine (PC) vesicles and their lipoplexes, on the complement system was studied using the method developed. It was revealed that PC vesicles did not affect the complement system, while CL vesicles manifested low activation. The influence of plasmid DNA and its lipoplex based on PC liposomes as well on the complement system was very low. PC/LacZ lipoplex (143 microg/ml) acted on the complement system like 5.36 microg/ml heat aggregated IgG (agg) (the level of no pathological ruptures), whereas CL/LacZ lipoplex (143 microg/ml) acted similar to 10.7 microg/ml IgG (agg). Thus, weak activation of the complement system with CL lipoplex, and even weaker for the PC lipoplex testified to the use of neutral and positively charged lipoplexes preferably in gene therapy protocols. The technique can also be used for testing the influence of injectable gene therapy vectors on the complement system.     

Stabilization of Diluted Aqueous Solutions of Horseradish Peroxidase

Effects of pH, enzyme concentration, and various supplements on the catalytic activity, temperature stability, and secondary structure of horseradish peroxidase (HRP) were studied in diluted aqueous solutions. In 5.0 mM citrate-phosphate buffer (pH 4.2) at 55°C and infinite dilution, HRP was inactivated with a rate constant of 2.86 × 10^–3 s^–1. CaCl₂, BSA, and glycerol caused protective effects, whereas KCl, LiCl, maltose, PEG-6000 (at a concentration above 3%), Triton X-100, ethanol, and Kathon CG had an opposite effect and altered the secondary structure of HRP. Two HRP-stabilizing media: the glycerol-based one containing 10% ethanol and 20% glycerol, or the protein-based one containing 0.1% Kathon CG and 0.2 mg/ml of BSA in 50.0 mM Tris-HCl buffer (pH 7.2) supplemented with 50 mM CaCl₂ were developed, and the stability of HRP (0.36 nM) and its immunoglobulin, cortisol, and progesterone conjugates were compared in these two media. The protein-based medium displayed a greater stabilizing effect particularly on HRP-steroid conjugates.