Role of methyl groups in dynamics and evolution of biomolecules

Recent studies have discovered strong differences between the dynamics of nucleic acids (RNA and DNA) and proteins, especially at low hydration and low temperatures. This difference is caused primarily by dynamics of methyl groups that are abundant in proteins, but are absent or very rare in RNA and DNA. In this paper, we present a hypothesis regarding the role of methyl groups as intrinsic plasticizers in proteins and their evolutionary selection to facilitate protein dynamics and activity. We demonstrate the profound effect methyl groups have on protein dynamics relative to nucleic acid dynamics, and note the apparent correlation of methyl group content in protein classes and their need for molecular flexibility. Moreover, we note the fastest methyl groups of some enzymes appear around dynamical centers such as hinges or active sites. Methyl groups are also of tremendous importance from a hydrophobicity/folding/entropy perspective. These significant roles, however, complement our hypothesis rather than preclude the recognition of methyl groups in the dynamics and evolution of biomolecules.     

Mitochondrial DNA haplogrouping of the brown bear,Ursus arctos (Carnivora: Ursidae) in Asia,based on a newly developed APLP analysis

Sequence analyses of the complete brown bear, Ursus arctos, mitochondrial DNA (mtDNA) genome have detected scattered single nucleotide polymorphisms (SNPs) that define distinct mtDNA haplogroups in phylogeographical studies. The degraded DNA in historical samples, such as stuffed or excavated specimens, however, is often not suitable for sequence analyses. To address this problem, we developed an amplified product length polymorphism (APLP) analysis for mtDNA‐haplogrouping U. arctos specimens by detecting haplogroup‐specific SNPs. We verified the validity and utility of this method by analysing up to 170‐year‐old skin samples from U. arctos specimens collected widely across continental Eurasia. We detected some of the same haplogroups as those occurring in eastern Hokkaido (Japan) and eastern Alaska in continental Eurasia (the Altai and the Caucasus). Our results show that U. arctos in eastern Hokkaido and eastern Alaska descended from a common ancestor in continental Eurasia, and suggest that U. arctos occupied several refugia in southern Asia during the Last Glacial Maximum. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 627–635.     

Purification, quaternary structure and immunological properties of phosphorylase kinase from chicken skeletal muscle

Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme as determined by gel filtration on Sepharose 4B is close to that of rabbit skeletal muscle phosphorylase kinase (i. e., approximately 1300 000). The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 140 000 beta, 129 000; gamma', 44 000; delta, 17 000 (cf. the Mr values of the alpha- and gamma-subunits of the rabbit muscle isoenzyme are 146 000 and 42 000). The four subunits, alpha', beta, gamma' and delta, were found to exist in equimolar amounts as shown by a densitometric analysis of acrylamide gels; hence, the subunit formula of the chicken skeletal muscle isoenzyme is (alpha' beta gamma' delta)4. Rabbit antisera against a mixture of alpha'- and beta-subunits of chicken phosphorylase kinase yield a single precipitin line with this enzyme, do not show cross reactions of identity with the rabbit muscle enzyme but strongly inhibit the activity of the chicken enzyme and partially inhibit the activity of the rabbit muscle isoenzyme.     

Improved Dot-Blot Hybridization Assay for Large-Scale Detection of Potato Viruses in Crude Potato Tuber Extracts

Abstract A rapid and simple technique has been developed to enhance the sensitivity of virus detection in dot-blot hybridization assay by up to 1000 fold. The procedure generally follows that of B aulcombe et al. (1984) but includes moderate heating of the nitrocellulose filter in 10XSSC, 0.5% SDS solution at 55°C after sample application. Using this procedure, four potato viruses (PVX, PVS, PVM and PVY) were detected with cloned virus-specific ³²P-cDNA probes in 2 μl spots containing 0.2–2 pg of purufied virus (0.1–1 ng/ml). The procedure was also successfully applied for the detection of PVX, PVS, PVM and PVY in crude potato tuber extracts.     

Chemico-enzymatic synthesis and cloning of human angiogenin gene in M13mp8 phage

The nucleotide sequence coding for human angiogenin has been deduced from the published amino acid sequence with the use of codons preferentially utilized in highly expressed E. coli genes. It was divided into forty-three oligonucleotides, which were synthesized by automatic gene assembler and then joined by DNA ligase into three double-stranded blocks, the blocks were consequently cloned and ligated in M13mp8 phage, and the resultant 389-bp DNA sequence coding for human angiogenin was analysed by chain-terminator sequencing technique.     

Structural and functional analysis of the respiratory burst in the phagocytosing macrophage

The interrelation between structural changes and oxygen consumption by the phagocyting macrophage was studied. The mean number of phagocyted particles was estimated by the method of stereological transformation. It is found that the uptake of yeast particles and CN- -nonsensitive oxygen consumption is related to the concentration of yeast cells in the incubation medium. A positive correlation was established between the oxygen consumption and the mean number of phagocyted particles. The results obtained may suggest that the "respiration burst" takes place in the contact area of the macrophage and the phagocyted material, and its extent probably depends on the surface of that contact area.     

A-like cells of the gastric mucosa as a possible source of prostaglandins

AL cells of the oxyntic stomach area were studied in rats using ultrastructurometric technique. High-threshold, short-term direct electrical vagostimulation (5 V, 4 msec, 30 Hz, 10 sec) was performed in experimental group of 12 animals. Animals were killed 1, 10 and 30 min after stimulation. High post-stimulation lipolytic activity of AL cells and intensification of "granule autophagy" phenomenon were noted. Our findings as well as the literature data suggest a hypothesis on possible prostaglandin production by AL cells. Direct evidence in favour of this hypothesis is difficult to obtain due to the lack of sufficiently reliable methods of their morphological detection in cells.     

Immunoenzyme detection of specific brain antigens as a criterion of the permeability of the hemato-encephalic barrier in rats following acute gamma irradiation

The immunoenzyme detection systems for the measurement of the alpha-2 globulin of the brain (alpha 2M) and glial fibrillary acidic antigens (GFAP) were developed. These systems were used for the study of the penetration through hemato-encephalic barrier in rats subjected to gamma radiation. This method is recommended for the indirect evaluation of the hemato-encephalic barrier functional disorders.