Recognition and use of the unusual X-DNA as a primer-template by Klenow DNA polymerase enzyme

Based on CD spectra, 2-amino-2'-deoxyadenosine-containing synthetic alternating DNA, poly(amino2dA-dt) undergoes a conformational transition from a B-form to a non-Z zig-zag form of DNA, called X, even under conditions where enzymes can work. Kinetic parameters of the E. coli Klenow DNA polymerase enzyme-catalyzed copying of both the B- and X-forms of poly(amino2dA-dT) have been determined. Binding affinity of X-DNA to the enzyme proved to be even higher than that of the B-DNA; primer-chain extension of X-poly(amino2dA-dT) was however hindered as compared to its B-form. This differential utilization of X-DNA versus B-DNA by a DNA polymerase is an in vitro enzymatic evidence of an unusual DNA conformation.     

Transformation of erythrocytes and their ultrastructure with respect to aging

Transformation and ultrastructure of erythrocytes have been investigated in 58 white non-inbred rats on the 1st, 2nd, 3d days after birth and at the age of 1.5, 3, 8, 20 months. The transformation index of the erythrocytes drops by the 8th month of life and further it rises in old animals. In newborn rats echino-poikiloid forms predominate; they are mainly presented as reticulocytes with remnants of nuclei, mitochondria and other organells. During first three days of life cells of the erythroid line actively free themselves from the nucleus by means of its pyknosis and chromatinolysis. In newborn rats erythrocytes have folds of plasmolemma of linear and spotted form on their surface; their number sharply decreases in mature animals. The least changes in form and ultrastructure of erythrocytes are noticed in 3-8-month-old animals.     

Chromatin matrix activity in hepatocyte nuclei after disturbance of the hepatic vagal innervation

The level of matrix activity of extranucleolar and nucleolar chromatin in hepatocytes of white noninbred rats has been studied in dynamics after disturbance of the vagal innervation. In order to estimate the labelling intensity of nuclear structures, the histoautoradiographic method is used. After bilateral subdiaphragmal vagotomy at early stages (1-2 weeks) the level of the nucleoplasm and nucleolus increases essentially, and by the 30th day a tendency to its normalization is noted. A conclusion is made that the peripheral nervous system influences the intensity of metabolic processes in the nucleus.     

Vimentin-positive epithelial cells in aggregated lymphoid nodules (Peyer's patches) in rabbits

Peculiarities of cytoskeleton in membranous cells and disposition of the latter in the cupola epithelium in aggregated lymphoid nodules++ (ALN) have been studied in the ileum of 5 rabbits. The material has been fixed in liquid nitrogen and in the mixture of paraformaldehyde and glutar aldehyde. Methods of immunomorphology, high resolving light and transmissive electron microscopy have been used. Monoclonal antibodies to vimentin are selectively bind with a specific population of the ALN cupola epithelial cells. These cells are regularly arranged in the epithelium of the cupola lateral part and they are absent in the epithelium of the intestinal crypts, villi and apex of the cupolas. In the lateral epithelium of the cupolas surface, nearer to their base vimentin-positive++ epitheliocytes make contacts with single interepitheliocytic lymphocytes, and nearer to the apex they surround compact groups of the interepitheliocytic lymphocytes. The vimentin-positive++ epitheliocytic cells are identified as M-cells.     

Changes in the cellular composition of the deciduous membrane in physiologic pregnancy and late toxicosis

Cytochemical characteristics of the decidual membrane at a physiologically normal pregnancy and in cases of late toxicosis are presented. Three main cell types of the decidual membrane are defined: large decidual cells (LDC), small decidual cells (SDC) and granular cells of endometrium, or K-cells. Part of each cell type in the decidual membrane is determined. At physiologically normal pregnancy the part of the LDC makes 50-60% in the membranes and 80-85% in the basal plate of the placenta; SDC--10-18% in the fetal membranes and 1-2% in the basal plate of the placenta; K-cells--0.5-1%. At late toxicosis of pregnancy there is a change in relative and absolute amount of the decidual cells: the part of the LDC decreases up to 26-40% in the fetal membranes and up to 55% in the basal plate of the placenta; part of the K-cells at a slight form of preeclampsia rises up to 3-4%, at a severe form--up to 11-12%. The change in cell composition results in certain disturbances of physiological equilibrium of biologically active substances produced by the decidual cells. This correlates with the severity and clinical manifestations of the late toxicosis of pregnancy. Correlation of the decidual cells disfunction, directed to regulation of their reproduction and functioning, can become one of the elements of pathogenic treatment of the late toxicosis of pregnancy.     

Non-C-G recognition sequences of DNA cytosine-5-methyltransferase from rat liver

The eukaryotic DNA cytosine-5-methyltransferase (E.C.2.1.1.37) is known to methylate cytosine in DNA mainly, but not exclusively in C-G. In the present study the minor, non-C-G recognition sequences of a rat DNA methyltransferase were analyzed by Maxam-Gilbert sequencing of in vitro methylated SV40 DNA. The enzyme methylates C-A and C-T at a 50-fold lower initial rate than C-G. Methylation of C-C at the 5'C was not observed in the piece of DNA sequenced. The methylation of C-A is very low in the trinucleotides ACA and CAC, the other C-A containing trinucleotides in DNA are much better methylacceptors. C-T was found methylated predominantly in the sequences CCTAA, ACTAA, and ACTGT. A comparison of the activity with different substrates is in favour of the enzyme making its recognition in the major groove of the DNA.     

Hydrodynamic and circular dichroic analysis of mammary-derived growth inhibitor (MDGI)

The mammary-derived growth inhibitor exists in solution as a monomeric molecule with a molar mass of 14,500 +/- 400 g/mol. The largest diameter and the height of the polypeptide chain were estimated to be 3.75 +/- 0.25 nm and 2.01 +/- 0.13 nm respectively. This is in good agreement with the structurally related bovine peripheral myelin P2 protein (about 70% amino acid sequence homology). CD measurements have revealed MDGI to be a protein with about 50% beta structure and less than 20% alpha helix similarly as in fatty acid-binding proteins. Removal of endogenous long-chain fatty acid by lipidex or storage in the frozen state lead to a destabilization of the active MDGI conformation which is accompanied by a loss of its activity with regard to growth inhibition of Ehrlich Ascites cells.     

Modulation of mitochondrial F0F1 catalysis by boundary and bulk phase phospholipids

The importance of boundary and bulk phase phospholipids was studied on a mitochondrial ATPase complex isolated by AH-Sepharose chromatography as described by Dreyfus et al (1984, Anal. Biochem. 142,215-220), this preparation was devoid of the adenine nucleotide carrier. The presence of isoelectric or acidic phospholipids during the purification in the column allows the exchange of tightly bound phospholipids up to 95%. ATP hydrolysis and oligomycin sensitivity were slightly affected by the nature of boundary and bulk phase phospholipids, while Pi-ATP exchange was highly inhibited.     

The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3

The ras-related rho A protein expressed in E. coli, was ADP-ribosylated by botulinum ADP-ribosyltransferase C3. C3 also modified the valine-14 mutant rho protein but not the products of H-ras, R-ras, ral, ypt, and rap 1 genes. A ras-rho chimaera consisting of 60 amino acids from the amino terminus of ras fused to 133 amino acids from the carboxy terminus of rho was not modified by C3. Antibodies raised against the porcine brain cytosolic substrate of C3 cross reacted with the rho, valine-14 rho and ras-rho proteins, but not with the gene products of H-ras, R-ras, ral or rap 1. Polyclonal anti-H-ras antibodies cross reacted with H-ras but not with ral, rho, or the C3 substrate purified from porcine brain.     

Comparison of polymerase insertion and extension kinetics of a series of O2-alkyldeoxythymidine triphosphates and O4-methyldeoxythymidine triphosphate

The effect of alkyl group size on ability to act as deoxythymidine triphosphate (dTTP) has been studied for the carcinogen products O2-methyl-, O2-ethyl-, and O2-isopropyl-dTTP by using three types of nucleic acids as template and DNA polymerase I (Pol I) or Klenow fragment as the polymerizing enzymes. Apparent Km and relative Vmax values were determined in primer extension on M13 DNA at a single defined site, in poly[d(A-T)], and in nicked DNA. These data are the basis for calculation of the relative rate of insertion opposite A, relative to dTTP. The insertion rate for any O2-alkyl-dTTP is much higher than for a mismatch between unmodified dNTPs. Unexpectedly, O2-isopropyl-dTTP is more efficiently utilized than O2-methyl-dTTP or O2-ethyl-dTTP on each of the templates. O2-isopropyl-dTTP also substitutes for dTTP over extended times of DNA synthesis at a rate only slightly lower than that of dTTP. Parallel experiments using O4-methyl-dTTP under the same conditions show that it is incorporated opposite A more frequently than is O2-methyl-dTTP. Therefore, both the ring position and the size of the alkyl group influence polymerase recognition. Once formed, all O2-alkyl-T.A termini permit elongation, as does O4-methyl-T.A. In contrast to the relative difficulty of incorporating the O-alkyl-dTTPs, formation of the following normal base pair (C.G) occurs rapidly when dGTP is present. This indicates that a single O-alkyl-T.A pair does not confer significant structural distortion recognized by Pol I.