Synthesis and physical characterization of DNA fragments containing N4-methylcytosine and 5-methylcytosine.

The synthesis of N4-methyl-2'-deoxycytidine and its fully protected mononucleotide, suitable for the oligonucleotide synthesis by phosphotriester method is described. A set of octanucleotides - d(CGCGCGCG), d(CG5mCGCGCG), d(CG4mCGCGCG) and dodecanucleotides - d(GGACCCGGGTCC), d(GGA5mCCCGGGTCC), d(GGA4mCCCGGGTCC) has been synthesized in a solution. Physical characterization of the oligonucleotide duplexes by means of UV and CD spectrometry provides the evidence that 4mC similarly to 5mC favours the B--greater than Z transition, although both of these methylated cytosines inhibit the B--greater than A conformational change. N4-Methylcytosine in contrast to 5-methylcytosine reduces the DNA double helix thermal stability.     

Sequence motifs characteristic of DNA[cytosine-N4]methyltransferases: similarity to adenine and cytosine-C5 DNA-methylases

The sequences coding for DNA[cytosine-N4]methyltransferases MvaI (from Micrococcus varians RFL19) and Cfr9I (from Citrobacter freundii RFL9) have been determined. The predicted methylases are proteins of 454 and 300 amino acids, respectively. Primary structure comparison of M.Cfr9I and another m4C-forming methylase, M.Pvu II, revealed extended regions of homology. The sequence comparison of the three DNA[cytosine-N4]-methylases using originally developed software revealed two conserved patterns, DPF-GSGT and TSPPY, which were found similar also to those of adenine and DNA[cytosine-C5]-methylases. These data provided a basis for global alignment and classification of DNA-methylase sequences. Structural considerations led us to suggest that the first region could be the binding site of AdoMet, while the second is thought to be directly involved in the modification of the exocyclic amino group.     

An improved method for the detection of Dcm methylation in DNA molecules

The intrinsic insensitivity of EcoRII recognition sites in RF DNAs of phage M13 and vector M13mp18 towards this restriction endonuclease can be overcome by adding site-specific oligodeoxyribonucleotide duplexes to the restriction sample. Since Dcm- DNA but not Dcm(+)-methylated DNA becomes susceptible under these conditions, this procedure constitutes an improvement of the Dcm methylation assay.     

Investigation of dye/protein interaction and its application to enzyme purification

In this review the results of the interaction of the active dyes used in the USSR textile industry with microbial enzymes and blood serum proteins are discussed. The complexity of dye/protein interaction and the dependence of this interaction on different factors is demonstrated. Some practical aspects of the use of dye containing sorbents are presented and discussed. Their suitability for RNA ligase and DNA ligase, acetate kinase, alcohol dehydrogenase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase purification and blood serum protein fractionation is demonstrated.     

Structural and functional studies of the adrenal zona glomerulosa in sodium-depleted and sodium-loaded sheep

Cell and Tissue Research - The effects of alterations in sodium status upon the morphology of the adrenal zona glomerulosa in sheep have been examined qualitatively and quantitatively, using...     

Site-specific endonucleases LplI and AagI

New site-specific endonucleases LplI and AagI have been isolated from the Lactobacillus plantarum and Achromobacter agile cells, respectively. The enzymes' purification stages included treatment of cell-free extracts with polyethylenimine, fractionation in two-phase system by Albertsson's method, chromatography on blue Sepharose and DEAE-cellulose. The results of cleavage of a 5'-32P-labelled oligodeoxynucleotide duplex by restriction endonucleases LplI and AagI indicate that these enzymes recognize and cut the sequence AT decreases CGAT, being therefore true isoschizomers of the ClaI restriction endonuclease from Caryophanon latum. The L. plantarum strain has 400 fold endonuclease productivity as compared with the ClaI producent and is perspective for preparative isolation of LplI.     

The morphofunctional transformation of skeletal muscle as a consequence of electroneurostimulation training]

By means of histological and histochemical methods slices of biopsies of the canine musculus latissimus dorsi have been investigated after electroneurostimulation for three months through the thoracodorsal nerve in situ and after cutting its initial part. Frequency of contractions increases gradually from 30 up to 80 per 1 min every 2 weeks. The preparations are stained with hematoxylin--eosin. Histochemical reactions for adenosinetriphosphatase (ATPase) (incubational medium pH 4.3 and 10.3) and for acetylcholinesterase (AChE) are performed. The muscle, stimulated in situ, preserves its normal structure. Transformation of muscle contractions from fast to slow, as well as increasing AChE activity in myoneural synapses are revealed. The muscle, stimulated after its cutting at its beginning, is subjected to adipose dystrophy. Activity of ATPase and AChE do not differ from that in the control.     

Selecting neural networks for a committee decision

To improve recognition results, decisions of multiple neural networks can be aggregated into a committee decision. In contrast to the ordinary approach of utilizing all neural networks available to make a committee decision, we propose creating adaptive committees, which are specific for each input data point. A prediction network is used to identify classification neural networks to be fused for making a committee decision about a given input data point. The jth output value of the prediction network expresses the expectation level that the jth classification neural network will make a correct decision about the class label of a given input data point. The proposed technique is tested in three aggregation schemes, namely majority vote, averaging, and aggregation by the median rule and compared with the ordinary neural networks fusion approach. The effectiveness of the approach is demonstrated on two artificial and three real data sets.     

Influence of surface characteristics on the stability of Cryptosporidium parvum oocysts

Microelectrophoresis is a common technique for probing the surface chemistry of the Cryptosporidium parvum oocyst. Results of previous studies of the electrophoretic mobility of C. parvum oocysts in which microelectrophoresis was used are incongruent. In this work we demonstrated that capillary electrophoresis may also be used to probe the surface characteristics of C. parvum oocysts, and we related the surface chemistry of C. parvum oocysts to their stability in water. Capillary electrophoresis results indicated that oocysts which were washed in a phosphate buffer solution had neutrally charged surfaces. Inactivation of oocysts with formalin did not influence their electrophoretic mobility, while oocyst populations that were washed in distilled water consisted of cells with both neutral and negative surface charges. These results indicate that washing oocysts in low-ionic-strength distilled water can impart a negative charge to a fraction of the oocysts in the sample. Rapid coagulation experiments indicated that oocysts did not aggregate in a 0.5 M NaCl solution; oocyst stability in the salt solution may have been the result of Lewis acid-base forces, steric stabilization, or some other factor. The presence of sucrose and Percoll could not be readily identified on the surface of C. parvum oocysts by attenuated total reflectance-Fourier transform infrared spectroscopy, suggesting that these purification reagents may not be responsible for the stability of the uncharged oocysts. These findings imply that precipitate enmeshment may be the optimal mechanism of coagulation for removal of oocysts in water treatment systems. The results of this work may help elucidate the causes of variation in oocyst surface characteristics, may ultimately lead to improved removal efficiencies in full-scale water treatment systems, and may improve fate and transport predictions for oocysts in natural systems.