N-acetylcysteine-induced changes in susceptibility of transformed eukaryotic cells to bacterial invasion

The effect of N-acetylcysteine (NAC) on morphological and physiological properties of "normal" 3T3 and 3T3-SV40 fibroblasts was studied. Incubation of the cells with 10 and 20 mM NAC for 20 h resulted in a reversible increase in the intracellular level of reduced glutathione and disorganization of actin cytoskeleton. Surprisingly, upon removal of NAC, 3T3-SV40 fibroblasts demonstrated formation of well-adhered cells with structured 3T3-like stress-fibers. Neither changes in glutathione levels, nor cytoskeleton disorganization/assembly abolished resistance of 3T3 cells to invasion by the bacterium Escherichia coli A2. On the other hand, pretreatment with NAC converted bacteria-susceptible 3T3-SV40 cells into resistant ones. These results show that NAC can induce partial reversion of transformed phenotype. We suggest that this effect is due to NAC-induced modifications of cell surface proteins rather than to changes in the level of intracellular glutathione.     

Mitochondrial NADH fluorescence is enhanced by complex I binding

Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several-fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10-fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state.     

Galleria mellonella larvae fat body disruption (Lepidoptera: Pyralidae) caused by the venom of Habrobracon brevicornis (Hymenoptera: Braconidae)

The ability of Habrobracon brevicornis venom to elevate the nutritional suitability of a host by affecting the host larvae fat body condition was studied. To understand whether H. brevicornis crude venom impacts the host biochemical profile, the concentrations of total lipids and main sugars in the host larvae lymph were analyzed. All measurements were carried out during the first 3 days after envenomation. A significant increase in the lipid level was fixed only on the second day after envenomation. A significant increase in the total trehalose count was detected during all 3 days, while a significant increase in glucose concentration was noted only on the first day. Well‐observed disruptions were fixed in thin and semithin sections of the G. mellonella larval fat body starting from the second day after envenomation. Significant increases in both phospholipase A₂ and C enzyme activity as well as acid proteases were detected in the wax moth fat body after envenomation during all experimental times. At the same time, imbalances in the antioxidant system, including changes in the activities of superoxide dismutase, peroxidases, catalase, and glutathione‐S‐transferase, were detected. The reliable increase in the expression of the gene encoding Hsp70 was fixed both for 24 and 48 h after envenomation, while a reliable increase in the expression of the gene encoding inhibitor of apoptosis protein was detected only 24 h after wax moth larvae envenomation. Considering the absence of DNA fragmentation, the imbalance in the “ROS/antioxidants” system, and the increased activity of phospholipases and acid proteases in the fat body cells from envenomated wax moth larvae, we can hypothesize that the fat body disruption occurs in a necrotic manner. The results of the work expand the knowledge about the biochemical aspects of interaction between ectoparasitoids and their hosts     

The use of theVibrio harveyi luminescence mutagenicity assay as a rapid test for preliminary assessment of mutagenic pollution of marine sediments

Mutagenic pollution of the natural environment is currently one of the most serious environmental problems. It includes the pollution of marine sediments. Therefore, rapid detection of the presence of mutagens is an important issue. Recently, we have developed a novel microbiological assay for rapid assessment of mutagenicity of samples from the natural environment. This assay is based on bioluminescence of a mutant Vibrio harveyi strain, and was shown to be useful in testing samples of marine water and plant tissues. Here we demonstrate the usefulness of this assay in preliminary assessment of mutagenic pollution of marine sediments. Mutagenicity of environmental samples taken from the Baltic Sea, is documented and compared here with a commercially available standard sediment sample (IAEA 383), which contains known amounts of mutagenic compounds. The whole procedure, from obtaining a sample in the laboratory to getting final results, is very short (less than 4 h).     

Further studies on water mites from Korea,with description of two new species (Acari,Hydrachnidia)

New records of water mites (Acari: Hydrachnidia) from streams in South Korea are presented. Two species are described as new to science: Torrenticola neodentifera sp. n. (Torrenticolidae) and Atractides ermilovi sp. n. (Hygrobatidae). Five species are reported as first records for Korea: Wandesia (Wandesia) reducta Tuzovskij, 1987, Wandesia (Wandesia) cf. rara Tuzovskij, 1990, Sperchon (Sperchon) orientalis Tuzovskij, 1990, Feltria (Feltria) kuluensis Tuzovskij, 1988 and Atractides (Atractides) constrictus (Sokolow, 1934). The latter species is redescribed and elevated to species rank based on new material from the Russian Far East.     

Proteogenomics meets cancer immunology: mass spectrometric discovery and analysis of neoantigens

Cancer proteogenomics is an emerging field that aims to identify and quantify protein sequence changes associated with the cancer genome. Besides being involved in cancer development and progression, such protein variants may serve as neoantigens, which provide the T-cell response against tumors. Mass spectrometry-based proteogenomics may be a promising tool for finding neoantigens in individual specimens. It is partly based on a technical background accumulated from mass spectrometric studies of peptide ligands of major histocompatibility complex proteins. Examples of the use of mass spectrometry in neoantigen identification are reviewed in this article. Some experimental workflows are discussed, which may use shotgun and targeted proteomics for translational human studies of neoepitopes, such as cancer vaccine development and checkpoint therapy response prediction.     

Derivation of Hair-Inducing Cell from Human Pluripotent Stem Cells

Dermal Papillae (DP) is a unique population of mesenchymal cells that was shown to regulate hair follicle formation and growth cycle. During development most DP cells are derived from mesoderm, however, functionally equivalent DP cells of cephalic hairs originate from Neural Crest (NC). Here we directed human embryonic stem cells (hESCs) to generate first NC cells and then hair-inducing DP-like cells in culture. We showed that hESC-derived DP-like cells (hESC-DPs) express markers typically found in adult human DP cells (e.g. p-75, nestin, versican, SMA, alkaline phosphatase) and are able to induce hair follicle formation when transplanted under the skin of immunodeficient NUDE mice. Engineered to express GFP, hESC-derived DP-like cells incorporate into DP of newly formed hair follicles and express appropriate markers. We demonstrated that BMP signaling is critical for hESC-DP derivation since BMP inhibitor dorsomorphin completely eliminated hair-inducing activity from hESC-DP cultures. DP cells were proposed as the cell-based treatment for hair loss diseases. Unfortunately human DP cells are not suitable for this purpose because they cannot be obtained in necessary amounts and rapidly loose their ability to induce hair follicle formation when cultured. In this context derivation of functional hESC-DP cells capable of inducing a robust hair growth for the first time shown here can become an important finding for the biomedical science.