Characterization of CoCas9 nuclease from Capnocytophaga ochracea.

Cas9 nucleases are widely used for genome editing and engineering. Cas9 enzymes encoded by CRISPR-Cas defence systems of various prokaryotic organisms possess different properties such as target site preferences, size, and DNA cleavage efficiency. Here, we biochemically characterized CoCas9 from Capnocytophaga ochracea, a bacterium that inhabits the oral cavity of humans and contributes to plaque formation on teeth. CoCas9 recognizes a novel 5'-NRRWC-3' PAM and efficiently cleaves DNA in vitro. Functional characterization of CoCas9 opens ways for genetic engineering of C. ochracea using its endogenous CRISPR-Cas system. The novel PAM requirement makes CoCas9 potentially useful in genome editing applications.

The Profile of Post-translational Modifications of Histone H1 in Chromatin of Mouse Embryonic Stem Cells.

Linker histone H1 is one of the main chromatin proteins which plays an important role in organizing eukaryotic DNA into a compact structure. There is data indicating that cell type-specific post-translational modifications of H1 modulate chromatin activity. Here, we compared histone H1 variants from NIH/3T3, mouse embryonic fibroblasts (MEFs), and mouse embryonic stem (ES) cells using matrix-assisted laser desorption/ ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS). We found significant differences in the nature and positions of the post-translational modifications (PTMs) of H1.3-H1.5 variants in ES cells compared to differentiated cells. For instance, methylation of K75 in the H1.2-1.4 variants; methylation of K108, K148, K151, K152 K154, K155, K160, K161, K179, and K185 in H1.1, as well as of K168 in H1.2; phosphorylation of S129, T146, T149, S159, S163, and S180 in H1.1, T180 in H1.2, and T155 in H1.3 were identified exclusively in ES cells. The H1.0 and H1.2 variants in ES cells were characterized by an enhanced acetylation and overall reduced expression levels. Most of the acetylation sites of the H1.0 and H1.2 variants from ES cells were located within their C-terminal tails known to be involved in the stabilization of the condensed chromatin. These data may be used for further studies aimed at analyzing the functional role played by the revealed histone H1 PTMs in the self-renewal and differentiation of pluripotent stem cells.

[Recombinant small heat shock protein from Acholeplasma laidlawii increases the Escherichia coli viability in thermal stress by selective protein rescue].

In both prokaryotes and eukaryotes, the survival at temperatures considerably exceeding the optimum is supported by intense synthesis of the so-called heat shock proteins (HSPs), which act to overcome the adverse effects of heat stress. Among mycoplasmas (class Mollicutes), which have significantly reduced genomes, only some members of the Acholeplasmataceae family possess small HSPs of the α-crystallin type. Overproduction of a recombinant HSP IbpA (Hsp20) from the free-living mycoplasma Acholeplasma laidlawii was shown to increase the resistance of Escherichia coli to short-term heat shock. It has been long assumed that IbpA prevents protein aggregation and precipitation thereby increasing viability of E. coli cells. Several potential target proteins interacting with IbpA under heat stress were identified, including biosynthetic enzymes, enzymes of energy metabolism, and components of the protein synthesis machinery. Statistical analysis of physicochemical properties indicated that IbpA interaction partners significantly differ in molecular weight, charge, and isoelectric point from other members of the E. coli proteome. Upon shortterm exposure to increased temperature, IbpA was found to preferentially interact with high-molecular weight proteins having a pI of about 5.1, significantly lower than the typical values of E. coli proteins.

THE SHORTENED ISOFORM OF a-TUBULIN IS DETECTED IN COMPLEX WITH PROTEASOMES.

The proteasome is a multi-subunit protein complex that serves as a major pathway for intracellular protein degradation playing important functions in various biological processes. By using MALDI-ICR-mass-spectrometry and Western-blot analysis, we have shown the presence of shortened isoform of a-tubulin in complex with the affinity-purified proteasomes from stable cell lines K562 and HEK293.

Experimental model of osteonecrosis of the jaw in rats treated with zoledronic acid.

We have examined the development of medication-related osteonecrosis of the jaws (MRONJ) in rats with no previous accumulation of zoledronic acid in the mandible. Ten male Wistar rats (weight 350-400g) were anaesthetised with chloral hydrate 450mg/kg intraperitoneally and the first and second mandibular molars on the left side were extracted. The five experimental rats were given six injections of zoledronic acid 0.18mg/kg over the next four weeks (total dose 1.08mg/kg). Two injections were given at once as an intravenous bolus injection (0.36mg/kg). Then rats were given 4 injections (0.18mg/kg) with 1 week interval over the next four weeks, after which they observed for a further four weeks. The five control rats were injected with saline. At the end of the eighth week, the animals were killed by asphyxiation in a carbon dioxide chamber, and their bone structure was visualised using cone-beam computed tomography (CT) and Galaxis software. We then studied the mandibles histopathologically to investigate the incidence of necrosis and infiltration of inflammatory cells. The cone-beam CT images in the experimental group showed deficiencies in the bone structure in the extracted molar area of the lower alveolar ridge. The histological findings in the mandibles of the group given zoledronic acid showed necrosis and infiltration of inflammatory cells, which were not present in the control group. We conclude that the immediate effect of zoledronic acid on the bone tissue during regeneration is an important factor in the development of MRONJ, in addition to the previously reported effects of the duration of treatment with zoledronic acid.

Post-translational modifications of linker histone H1 variants in mammals.

The covalent modifications of the linker histone H1 and the core histones are thought to play an important role in the control of chromatin functioning. Histone H1 variants from K562 cell line (hH1), mouse (mH1) and calf (cH1) thymi were studied by matrix-activated laser desorption/ionization fourier transform ion cyclotron resonance mass-spectroscopy (MALDI-FT-ICR-MS). The proteomics analysis revealed novel post-translational modifications of the histone H1, such as meK34-mH1.4, meK35-cH1.1, meK35-mH1.1, meK75-hH1.2, meK75-hH1.3, acK26-hH1.4, acK26-hH1.3 and acK17-hH1.1. The comparison of the hH1, mH1 and cH1 proteins has demonstrated that the types and positions of the post-translational modifications of the globular domains of the H1.2-H1.4 variants are very conservative. However, the post-translational modifications of the N- and C-terminal tails of H1.2, H1.3 and H1.4 are different. The differences of post-translational modifications in the N- and C-terminal tails of H1.2, H1.3 and H1.4 likely lead to the differences in DNA-H1 and H1-protein interactions.

[Mass spectrometric analysis of proteasomes affinity puirified from the human myelogenous leukemia cells K562].

Proteasomes carry out regulated proteolysis of most proteins and thereby play a crucial role in the regulation of different cellular processes. Dissecting subunit composition and post-translational modifications of proteasome is one of the important milestones in understanding their functions and mechanisms of regulation in the cell. To this end a strategy we followed a strategy for affinity purification of proteasomes from human myeloid leukemia cells with subsequent mass spectrometric analysis. Proteasomes were purified from the stable cell line K562 expressing HTBH tag-labeled 20S proteasome subunit ß7 (PSMB4) by non-covalent affinity purification on biotin-avidin beads, followed by elution with TEV protease. We identified all known subunits of the 26S proteasome, as well as PA200 and regulators PA28γ amongst the eluted proteins, using MALDI-ICR mass spectrometry. We have shown that the proteasomes are associated with heat shock proteins, components of the ubiquitin-proteasome system of some cytoskeleton proteins. A number of novel phosphorylation, ubiquitination and N-terminal modification of proteasome subunits were found for 16 proteasome subunits. Our results might be useful for further proteomic studies of proteasomes.

[The effect of red pigment of Saccharomyces cerevisiae on insulin fibril formation in vitro].

The effect of the yeast red pigment, the result of polymerization of AIR, and of its low molecular weight derivative (presumably devoid of phosphoribosyl moiety) on the formation of amyloid fibrils in vitro was studied. Both the red pigment and its derivative, the result of acid hydrolysis of the original pigment, were shown to diminish the intensity of amyloid bound Thioflavine T fluorescence. Correlation between the decrease of the intensity of Thioflavine T fluorescence and the concentration of both forms of the red pigment was demonstrated. Both forms were also able to compete with Thioflavine T for amyloid fibrils. Electron microscopy permitted to visualize a drop of fibril size in the case of red pigments presence during their formation.

[A simplified variant of the Maxam-Gilbert method for determining the primary structure of oligonucleotides and DNA fragments]. / Uproshchennyi variant metoda Maksama-Gilberta dlia opredeleniia pervichnoi struktury oligonukleotidov i fragmentov DNK.

A modification to the Maxam-Gilbert method is proposed that involves precipitation of the nucleotide material with the acetone solution of lithium perchlorate after the completion of chemical reactions to remove the reagents. Modification of cytosine residues is carried out in the presence of lithium chloride. The new mode of precipitation simplifies and speeds up the analysis of oligonucleotides and DNA fragments.