Interaction of a dimeric distamycin analog with poly(dA)poly(dT), poly[d(A–T)]poly[d(A–T)], and duplex O<Subscript>23</Subscript> at the origin of replication of the herpes simplex virus

The binding of a dimeric distamycin analog (Pt–bis–Dst) to poly[d(A–T)]poly[d(A–T)], poly(dA)poly(dT), and duplex O₂₃ with the sequence 5’-GCCAATATATATATATTATTAGG-3’, which occurs at the origin of replication (OriS) of the herpes simplex virus, was studied via UV and CD spectroscopy. The synthetic polyamide differs from the natural antibiotic in having two distamycin moieties that are linked via a glycine cis-diamino platinum group. The Pt–bis–Dst binding to poly[d(A–T)]poly[d(A–T)] and poly(dA)poly(dT) reached saturation at approximately one ligand molecule per eight bp. As the ligand–base pair ratio further increased, the maximum wavelength band tended to shift toward longer wavelengths in the CD spectra of complexes with poly[d(A–T)]poly[d(A–T)] and a shoulder appeared in the 290–310 nm spectral region that was absent from the CD spectra of complexes with lower ligand coverages. At higher ligand–oligonucleotide molar ratios, Pt–bis–Dst could bind to poly[d(A–T)]poly[d(A–T)] in the form of hairpins or associations that result from interactions between the distamycin moieties of two neighbor Pt–bis–Dst molecules. The structures of the complexes were stabilized by interactions between the pirrolcarboxamide moieties of two Pt–bis–Dst molecules absorbed on adjacent overlapping binding sites. The interactions could also be responsible for the concentration-dependent spectral changes that were observed during the formation of a complex between Pt–bis–Dst and poly[d(A–T)]poly[d(A–T)]. Spectral changes were almost absent in the case of Pt–bis–Dst binding to poly(dA)poly(dT). The binding of Pt–bis–Dst to duplex O₂₃ reached saturation at two ligand molecules per duplex, which contained a cluster of 18 AT pairs. At higher molar-concentration ratios, duplex CD spectra underwent changes similar to those that were observed for Pt–bis–Dst binding to poly[d(A–T)]poly[d(A–T)]. Testing Pt–bis–Dst for antiviral activity identified 1.5 μg/mL as a concentration that halved the cytopathic effect of the herpes simplex virus on Vero E6 cells; the selectivity index of antiviral action was 65; cytotoxicity was relatively low. The Pt–bis–Dst concentration that caused the death of approximately half of the cells was estimated at 100 μg/mL.     

Complexes of smooth muscle tropomyosin with F-actin studied by differential scanning calorimetry.

Differential scanning calorimetry (DSC) and light scattering were used to analyze the interaction of duck gizzard tropomyosin (tropomyosin) with rabbit skeletal-muscle F-actin. In the absence of F-actin, tropomyosin, represented mainly by heterodimers, unfolds at 41 degrees C with a sharp thermal transition. Interaction of tropomyosin heterodimers with F-actin causes a 2-6 degrees C shift in the tropomyosin thermal transition to higher temperature, depending on the tropomyosin/actin molar ratio and protein concentration. A pronounced shift of the tropomyosin thermal transition was observed only for tropomyosin heterodimers, and not for homodimers. The most pronounced effect was observed after complete saturation of F-actin with tropomyosin molecules, at tropomyosin/actin molar ratios > 1 : 7. Under these conditions, two well-separated peaks of tropomyosin were observed on the thermogram besides the peak of F-actin, the peak characteristic of free tropomyosin heterodimer, and the peak with a maximum at 45-47 degrees C corresponding to tropomyosin bound to F-actin. By measuring the temperature-dependence of light scattering, we found that thermal unfolding of tropomyosin is accompanied by its dissociation from F-actin. Thermal unfolding of tropomyosin is almost completely reversible, whereas F-actin denatures irreversibly. The addition of tropomyosin has no effect on thermal unfolding of F-actin, which denatures with a maximum at 64 degrees C in the absence and at 78 degrees C in the presence of a twofold molar excess of phalloidin. After the F-actin-tropomyosin complex had been heated to 90 degrees C and then cooled (i.e. after complete irreversible denaturation of F-actin), only the peak characteristic of free tropomyosin was observed on the thermogram during reheating, whereas the thermal transitions of F-actin and actin-bound tropomyosin completely disappeared. Therefore, the DSC method allows changes in thermal unfolding of tropomyosin resulting from its interaction with F-actin to be probed very precisely.     

Physiological analysis of the relation between the conditioned reflex and image memory in monkeys and preschool children

Interaction of short-term and long-term memory processes is subjected to phylo- and ontogenetic changes. Progressive development includes differentiation and independent manifestation of these memory forms together with formation of a more complex pattern of interaction between the latter.     

Mitochondrial Dysfunction Contributes to Oncogene-Induced Senescence

The expression of oncogenic ras in normal human cells quickly induces an aberrant proliferation response that later is curtailed by a cell cycle arrest known as cellular senescence. Here, we show that cells expressing oncogenic ras display an increase in the mitochondrial mass, the mitochondrial DNA, and the mitochondrial production of reactive oxygen species (ROS) prior to the senescent cell cycle arrest. By the time the cells entered senescence, dysfunctional mitochondria accumulated around the nucleus. The mitochondrial dysfunction was accompanied by oxidative DNA damage, a drop in ATP levels, and the activation of AMPK. The increase in mitochondrial mass and ROS in response to oncogenic ras depended on intact p53 and Rb tumor suppression pathways. In addition, direct interference with mitochondrial functions by inhibiting the expression of the Rieske iron sulfur protein of complex III or the use of pharmacological inhibitors of the electron transport chain and oxidative phosphorylation was sufficient to trigger senescence. Taking these results together, this work suggests that mitochondrial dysfunction is an effector pathway of oncogene-induced senescence.Mitochondria are central to cell metabolism and energy production. High-energy electrons coming from the oxidation of different carbon sources such as glucose and fatty acids enter the mitochondrial electron transport chain as reduced equivalents, and their energy gradually is converted into a proton gradient. Mitochondria use this gradient to synthesize ATP that later is used for biosynthetic reactions (9, 30). Mitochondria also control decisions for life and death. Changes in mitochondrial membrane permeability lead to the release of proapoptotic mediators that can kill cells with DNA damage or activated oncogenes (16). In this way, mitochondria control one of the major tumor suppressor responses: apoptosis (27). Some oncogenes, such as RasV12, STAT5, and Bcl2, have antiapoptotic activity, and some cell types have a high apoptosis threshold. Another tumor suppressor response, called cellular senescence, serves as a fail-safe mechanism against the transforming activity of antiapoptotic oncogenes (29, 40, 43). However, currently it is unknown whether mitochondria also can play a role in oncogene-induced senescence (OIS).OIS is phenotypically similar to the senescence response triggered by short telomeres, also known as replicative senescence (6). Replicative senescence is, in essence, the consequence of a DNA damage response triggered by short telomeres (11). OIS also involves the DNA damage response (2, 15, 28), but the mechanism of DNA damage and the contribution of mitochondria to it are unclear. It has been demonstrated that mitochondria play a critical role in replicative senescence, and several mitochondrial changes, including an increase in the production of reactive oxygen species (ROS), were reported in cells with short telomeres (34, 35). Mitochondrion-derived ROS contribute to the senescent phenotype by damaging the DNA (35) and therefore amplifying the DNA damage signals originally caused by short telomeres. We reasoned that a similar amplifying mechanism involving the mitochondria could operate in cells expressing oncogenes.Here, we use Ha-RasV12, an oncogenic allele of Ha-Ras, to study the role of mitochondria in OIS. RasV12 is a very important human oncogene and was the first linked to the senescence program (43). We report that oncogenic ras induces an increase in mitochondrial mass, mitochondrial DNA, and mitochondrial superoxide production before any sign of senescent cell cycle arrest. With time, these mitochondrial changes evolved into a severe mitochondrial dysfunction characterized by a further increase in ROS production, the accumulation of depolarized mitochondria around the cell nucleus, a decrease in ATP, and the activation of AMPK. The mechanism of the increase in mitochondrial mass and ROS in response to oncogenic ras was found to be dependent on either p53 or Rb. In addition, direct interference with mitochondrial functions by downregulating the mitochondrial Rieske iron sulfur protein (RISP) or by using pharmacological inhibitors of oxidative phosphorylation induced senescence. We suggest that the senescence effector mechanism acting downstream of p53 and Rb involves mitochondrial dysfunction.     

Organization of initial stages of somatic embryogenesis in tissue culture of Citrus sinensis cv. Tarocco at the organismal level

Four-step protocol was established for the in vitro regeneration of Citrus sinensis cv. Tarocco somatic embryos that were morphologically similar to small somatic embryos in vivo. The regeneration procedure comprises a mechanical destruction of embryogenic culture to obtain proembryogenic cell masses (PEMs) (step 1) followed by culturing on three different media (steps 2–4). The approach developed allows in vitro simulating somatic embryogenesis by dividing this process into three partially independent steps: PEM → globular somatic embryo → heart-shaped somatic embryo → somatic embryo with developed cotyledons. The highest frequency of morphogenetic stage transition was 64, 40, and 26%, respectively. It was shown that the first step (PEM → globular embryo) was associated with the formation of heterogeneous population of spherical bodies 50–500 μm in diameter, among which about 40% were somatic embryos at globular stage. The scheme is offered of alternative pathways for the development of spherical bodies in vitro, and interrelations between their sizes and ability to direct morphogenesis are discussed.     

Activation of mast cells after experimental myocardial infarction in 3 week-old rats

It is known that mast cells (MC) take an active part in regeneration processes in postinfarction heart in adult rats and humans. Behaviour of population of cardial MCs has been studied 20, 60, 75 and 90 days after experimental myocardial infraction induced in 3 week-old and adult rats by ligation of left coronary artery. The density of MC of different degrees of maturity was estimated in atrium and ventricle on paraffin sections stained with Alcian blue - Safranin. Findings were compared with MC density obtained in hearts of intact rats. The MC density in intact 1.5-2.5 month-old rats in atrium and ventricle was about 0.6 cells/mm2, in intact 3.5-4.0 month-old rats in atrium--1.2 cells/mm2, in ventricle--0.6 cells/mm2. The MC density in 3 week-old rats with infarction was significantly higher than in intact rats: 5-fold increase in 20 and 60 days in atrium, and 2-fold increase in 60 and 75 days in ventricle. In 60 days after infarction the MC density in adult rats was 3 times lower in atrium and 2 times lower in ventricle than in the same heart compartments of 3 week-old rats with infarction. After infarction in 3 week-old rats, a relative share of young cells with alcian-positive granules sharply increased in 20 days and then decreased by 60-75 days. This indicates a migration of immature MCs to infracted myocardium and their subsequent differentiation. The MC activation after infraction in young rats may result from a more active immune reaction in younger rats and/or functional peculiarities of their MC.