Electron microscope study of chromatin in nuclei of rat hepatocytes during age-related changes in the genome

On ultrathin liver sections, condensed chromatin of rat hepatocyte nuclei was studied. The animals were 2 days, 6 and 28 months old. It was established that neither maturation nor senescence were accompanied by the change of the relative square of total condensed chromatin. Relative square of perimembrane, nucleoplasmic and perinucleolar condensed chromatin were non changed either. Intensively proliferating hepatocytes of nascent animals were characteristic of maximal values of the following parameters (i) the relative length of the perimembrane condensed chromatin boundary with nucleoplasma. (ii) amount of chromatin clumps, (iii) the relative length of the nuclear membrane without condensed chromatin. For mature animals all these parameters are significantly decreased. For old rats as compared with mature ones the following parameters are significantly diminished: (i) the relative length of the perimembrane chromatin boundary with nucleoplasma, (ii) the relative length of the nuclear membrane without condensed chromatin, (iii) the mean square of the nucleolus. So, the known diminishing of the RNA synthesis at senescence is expressed morphologically in margination of condensed chromatin, in smoothing of the condensed chromatin surface responsible for the hnRNA synthesis and also in diminishing of the nucleolus responsible for the rRNA synthesis.     

Some properties of 1 P+f bacteriophage for Bacillus brevis var. G.-B and its nucleic acid]

The 1 P+f phage, a virulent mutant of the moderate P+ phage for Bac. brevis var. G.-B., consists of a hexagonal head (90x90 nm) and a long non-contractile tail (340 nm). This phage is characterized by a relatively long latent period (90-110 min) and a low yield (40-50 particles per cell). The 1P+f phage is quite stable at pH values from 1 to 11, insensitive to osmotic shock, treatment with chloroform and acridine orange. The sensitivity of the phage to thermal treatment and UV-radiation has been studied. The nucleic acid of the P+f phage is double-stranded DNA of AT-type (GC equals 34.5 mole %) which contains 5-methylcytosine (0.18 mole %) and N6-methyladenine (0.32 mole%). The level of methylation of cytosine and adenine residues in DNA of the 1 P+f phage does not depend on the host studied (Bac. brevis, P- and S variants). The specificity of methylation of cytosine residues in the S and P- cells appears to be the same. DNA of the 1 P+f phage strongly differs from DNA of the host in nucleotide composition (GC equals 45.7 mole %). Nevertheless, phage DNA is very similar to DNA from Bac. subtilis in the character of pyrimidine distribution (the amount of different pyrimidine isopliths). This may testify to a somewhat common character of the nucleotide sequence organization in DNA of the phage and its host.     

An update on the validation of whole slide imaging systems following FDA approval of a system for a routine pathology diagnostic service in the United States

Pathologists have used light microscopes and glass slides to interpret the histologic appearance of normal and diseased tissues for more than 150 years. The quality of both microtomes used to cut tissue sections and microscopes has improved significantly during the past few decades, but the process of rendering diagnoses has changed little. By contrast, major advances in digital technology have occurred since the introduction of hand held electronic devices, including the development of whole slide imaging (WSI) systems with software packages that can convert microscope images into virtual (digital) slides that can be viewed on computer monitors and via the internet. To date, however, these technological developments have had minimal impact on the way pathologists perform their daily work, with the exception of using computers to access electronic medical records and scholarly web sites for pertinent information to assist interpretation of cases. Traditional practice is likely to change significantly during the next decade, especially since the Federal Drug Administration in the USA has approved the first WSI system for routine diagnostic practice. I review here the development and slow acceptance of WSI by pathology departments. I focus on recent advances in validation of WSI systems that is required for routine diagnostic reporting of pathology cases using this technology.     

Effect of the antioxidant ionol on proteolytic apparatus of aging coleoptiles of wheat seedlings grown in light

The dynamics of changes in total proteolytic activity and activities of various groups of proteases in the coleoptiles of 3- to 12-day-old wheat seedlings grown in light with and without antioxidant BHT (2,6-di-tert-butyl-4-methylphenol) was studied. It was established that the specialized proteases that easily hydrolyze specific synthetic substrates and the enzymes actively hydrolyzing histone H1 dominate in young coleoptiles of 3- to 4-day-old seedlings. Proteases that degrade equally well the majority of the studied substrates are accumulated in the cells of old coleoptiles of 11- to 12-day-old seedlings. Under the effect of BHT, the plants grown in light (in comparison with etiolated seedlings) demonstrated a somewhat changed dynamics of proteolytic activity in young coleoptiles and the disappearance of proteases active toward histone H1. An inhibitory analysis revealed a relative domination of cysteine proteases in young coleoptiles at the initial development stage of seedlings, whereas the fraction of serine proteases markedly increased in old coleoptiles. We presume that the revealed quantitative and qualitative changes in the proteolytic apparatus of the coleoptile cells induced by BHT may be largely responsible for the retardant and geroprotective effect of this antioxidant in plants.     

The loss of CpC dinucleotides from DNA. II. Methylated and non-methylated genes of vertebrates

The frequencies of neighboring b.p. in more than 1100 genes of vertebrates in the EMBL bank (1000 kb) have been analysed. It has been found that the majority of such genes exhibit a lack of CpG duplexes and an excess of TpG+CpA. The loss of CpG may indicate that the major part of these sites in the genome is methylated and has been subjected to the pressure of CpG----TpG+CpA mutations. The methylated genes grouped into compartment M+ are represented by a fraction of repeated sequences and by genes of the most rapidly diverging families of proteins (globins, immunoglobulins, structural proteins, etc.). The genes of this compartment are characterized by a correlation between the G+C content and the value of CpG-suppression. A group of genes has been detected in which the CpG mutation process has gone so far that nearly all of these dinucleotides have disappeared from DNA. Judging by the value of CpG-suppression, these genes, grouped in the Mo+ compartment, used to be strongly methylated before. However, in the now extant vertebrates they have fully depleted their CpG reserve and for this reason lost the methylation capacity. Transitions in methylated CpG may be one of the sources of spontaneous mutagenesis resulting in the enhanced genetic instability of the cell. A gene compartment has been detected with an intermediate level of CpG deficiency; this compartment has been designated as M+. In these genes only a few of the available CpGs have been steadily methylated (and subjected to mutation). It has been found that the genome of vertebrates contains a specific CpG-rich fraction which exhibits no CpG-suppression, irrespective of the overall content of G+C. Probably, CpG sites have persisted unmethylated throughout the existence of these genes. We suggest them to constitute a M- compartment. This compartment comprises the genes of tRNA and rRNA (5S, 5.8S, 18S, 28S) and small nuclear RNAs U2-U6, as well as the genes of core histones, some enzymes, viruses and 5'-flanking sequences of certain protein-coding genes. In the genome of vertebrates, the genes of the evolutionary most conserved proteins and RNAs have not undergone methylation. A list of genes, belonging to different compartments of the vertebrate genome, is given. Compartment Mo+ constitutes 19%, M(+)--35%, M(+/-)--28% and M(-)--8% of all the vertebrate genes studied. Possible mechanisms, protecting the functionally most significant genes of vertebrates from methylation, and discussed.     

Specificity of methylation of cytosine risidues in DNA of Bacillus brevis var. G-B]

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of (methyl-3H)-methionine practically the total radioactivity included into DNA is found to exist in 5-methylcytosine (MC) and 6N-methyladenine (MA). The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of MC in Pur-MC-Pur and Pur-MC-T-Pur oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring with MC to be revealed and shows that MC localizes in G-MC-A and G-MC-T-Pu fragments. Bac. brevis S DNA-methylase modifying cytosine residues recognizes the GCAT GC degenerative nucleotide sequence which is a part of the following complementary structure with rotational symmetry: (5') ... N'--G--MC--T--G--C--N ... (3') (3') ... N--C--G--A--MC--G--N' ... (5') Cytosine modifying DNA-methylase activity is isolated from Bac. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence, DNA in bacterial cells can be partially undermethylated. This enzyme methylates cytosine residues in native and deneaturated DNA in the same nucleotide sequences. As compared to the native DNA, the denaturated DNA is indicative of a decrease in the level of methylation of adenine, rather than cytosine residues. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA (calf thymus, Pseudomonas aeruginosa etc.). DNA-methylases of different variants of Bac. brevis (R, S, P+, P-) methylate cytosine residues in the same nucleotide sequences. It means that specificity of methylation of DNA cytosine residues in the cells of different variants of Bac. brevis is the same.