Distribution of microtubules in prolactin cells of lactating rats

The intracellular distribution of microtubules was studied using serial sections of prolactin cells in anterior pituitary glands from lactating rats. Numerous microtubules were present in these cells following fixation with glutaraldehyde and osmium tetroxide. The greatest number of microtubules were present in the Golgi complex, situated around the perimeter and in association with the cisternae, vesicles and developing secretory granules. Microtubules were found in channels between groups of parallel cisternae of rough surfaced endoplasmic reticulum and in close proximity to small vesicles. They were also located adjacent to mitochondria, the plasmalemma, the nuclear envelope, and among mature secretory granules. Due to their orientation within the cell, it is suggested that the microtubules may act to direct the movement of organelles from one region of the cell to another and to give internal support to the cell.     

Repetitive DNA in and around translocation breakpoints of the Philadelphia chromosome

This paper describes systematic sequence studies of repetitive DNA in and around translocation breakpoints on chromosomes 9 and 22, which are involved in the formation of the Philadelphia chromosome in acute leukemias. In addition to Alu repeats described in previous studies, the breakpoint regions appear to contain many other repetitive elements, including a member of a new repetitive family (MER34) reported in this paper. Identification of these repeats broadens current studies on the possible involvement of repetitive DNA in this intensely studied chromosomal translocation.     

Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5′-Terminal Region of p53 mRNA In Vitro and in the Living Cells

The p53 protein is a key player in cell response to stress events and cancer prevention. However, up-regulation of p53 that occurs during radiotherapy of some tumours results in radio-resistance of targeted cells. Recently, antisense oligonucleotides have been used to reduce the p53 level in tumour cells which facilitates their radiation-induced apoptosis. Here we describe the rational design of antisense oligomers directed against the 5′-terminal region of p53 mRNA aimed to inhibit the synthesis of p53 protein and its ΔNp53 isoform. A comprehensive analysis of the sites accessible to oligomer hybridization in this mRNA region was performed. Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells. The antisense oligomers with 2′-OMe and LNA modifications were used to study the mechanism of their impact on translation. It turned out that the remaining RNase H activity of the lysate contributed to modulation of protein synthesis efficiency which was observed in the presence of antisense oligomers. A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins. Selected antisense oligonucleotides which were designed based on accessibility mapping of the 5′-terminal region of p53 mRNA were able to significantly reduce the level of p53 protein in MCF-7 cells. One of these oligomers might be used in the future as a support treatment in anticancer therapy.     

The Genetic Basis of Escherichia coli Pathoadaptation to Macrophages

Antagonistic interactions are likely important driving forces of the evolutionary process underlying bacterial genome complexity and diversity. We hypothesized that the ability of evolved bacteria to escape specific components of host innate immunity, such as phagocytosis and killing by macrophages (MΦ), is a critical trait relevant in the acquisition of bacterial virulence. Here, we used a combination of experimental evolution, phenotypic characterization, genome sequencing and mathematical modeling to address how fast, and through how many adaptive steps, a commensal Escherichia coli (E. coli) acquire this virulence trait. We show that when maintained in vitro under the selective pressure of host MΦ commensal E. coli can evolve, in less than 500 generations, virulent clones that escape phagocytosis and MΦ killing in vitro, while increasing their pathogenicity in vivo, as assessed in mice. This pathoadaptive process is driven by a mechanism involving the insertion of a single transposable element into the promoter region of the E. coli yrfF gene. Moreover, transposition of the IS186 element into the promoter of Lon gene, encoding an ATP-dependent serine protease, is likely to accelerate this pathoadaptive process. Competition between clones carrying distinct beneficial mutations dominates the dynamics of the pathoadaptive process, as suggested from a mathematical model, which reproduces the observed experimental dynamics of E. coli evolution towards virulence. In conclusion, we reveal a molecular mechanism explaining how a specific component of host innate immunity can modulate microbial evolution towards pathogenicity.     

Telocytes: new insight into the pathogenesis of gallstone disease

The major mechanisms of gallstone formation include biliary cholesterol hypersecretion, supersaturation and crystallization, mucus hypersecretion, gel formation and bile stasis. Gallbladder hypomotility seems to be a key event that triggers the precipitation of cholesterol microcrystals from supersaturated lithogenic bile. Telocytes, a new type of interstitial cells, have been recently identified in many organs, including gallbladder. Considering telocyte functions, it is presumed that these cells might be involved in the signalling processes. The purpose of this study was to correlate the quantity of telocytes in the gallbladder with the lithogenicity of bile. Gallbladder specimens were collected from 24 patients who underwent elective laparoscopic cholecystectomy for symptomatic gallstone disease. The control group consisted of 25 consecutive patients who received elective treatment for pancreatic head tumours. Telocytes were visualized in paraffin sections of gallbladders with double immunofluorescence using primary antibodies against c‐Kit (anti‐CD117) and anti‐mast cell tryptase. Cholesterol, phospholipid and bile acid levels were measured in gallbladder bile. The number of telocytes in the gallbladder wall was significantly lower in the study group than that in the control group (3.03 ± 1.43 versus 6.34 ± 1.66 cell/field of view in the muscularis propria, P < 0.001) and correlated with a significant increase in the cholesterol saturation index. The glycocholic and taurocholic acid levels were significantly elevated in the control subjects compared with the study group. The results suggest that bile composition may play an important role in the reduction in telocytes density in the gallbladder.     

Theoretical ab Initio Study of the Effects of Methylation on Structure and Stability of G:C Watson-Crick Base Pair

Abstract

Methylation of DNA occurs most readily at N(3), N(7), and O(6) of purine bases and N(3) and O(2) of pyrimidines. Methylated bases are continuously formed through endogenous and exogenous mechanisms. The results of a theoretical ab initio study on the methylation of G:C base pair components are reported. The geometries of the local minima were optimized without symmetry restrictions by the gradient procedure at DFT level of theory and were verified by energy second derivative calculations. The standard 6–31G(d) basis set was used. The single-point calculations have been performed at the MP2/6–31G(d,p), MP2/6–31₊₊G(d,p), and MP2/6–311₊₊G(2d,2p) levels of theory. The geometrical parameters, relative stability and counterpoise corrected interaction energies are reported. Also, using a variation-perturbation energy decomposition scheme we have found the vital contributions to the total interaction energy.     

Nonplanar DNA Base Pairs

Abstract

Three-dimensional structures of a representative set of more than 30 hydrogen-bonded nucleic acids base pairs have been studied by reliable ab initio quantum mechanical methods. We show that many hydrogen-bonded nucleic acid base pairs are intrinsically nonplanar, mainly due to the partial sp³ hybridization of nitrogen atoms of their amino groups and secondary electrostatic interactions. This finding extends the variability of intermolecular interactions of DNA bases in that i) flexibility of the base pairs is larger than has been assumed before, and ii) attractive proton-proton acceptor interactions oriented out of the base pair plane are allowed. For example, all four G…A mismatch base pairs are propeller twisted, and the energy preferences for the nonplanar structures range from less than 0.1 kcal/mol to 1.8 kcal/mol. We predict that nonplanarity of the amino group of guanine in the G(anti)…A(anti) pair of the ApG step of the d(CCAAGATTGG)₂ crystal structure is an important stabilizing factor that improves the energy of this structure by almost 3 kcal/mol. Currently used empirical potentials are not accurate enough to properly cover the interactions associated with amino-group and base-pair nonplanarity.