Role of telomerase in reactivation of macrophage nuclei in heterokaryons

It was shown that the duration of stay of macrophages in the peritoneal cavity of mice and method of their isolation did not affect markedly their capacity for resumption of DNA synthesis in heterokaryons. This means that mouse macrophage undergo such changes during differentiation that reactivation of DNA synthesis in their nuclei is only possible after interaction of telomeres with telomerase, since it was already shown that telomerase was involved in reactivation of DNA synthesis in the macrophage nuclei. The results of experiments did not reveal differences in the length of telomeres in mouse macrophages and other somatic cells. This could depend on the significant length of mouse telomeres and, as a result, their shortening, sufficient for the inhibition of proliferation, is beyond the limits of sensitivity of the current methods. It is also possible that changes in DNA properties in the macrophages occurring during their differentiation depend on changes in the conformation of the telomere complex in these cells. Testing of this suggestion is relevant with respect to recent data that cell hybridization, specifically in the form of heterokaryons, may be essential in realization of the therapeutic effect caused by the introduction of cells during cell therapy.     

The Effect of Azidothymidine on Reactivation of DNA Synthesis in Macrophage Nuclei Contained in Heterokaryons

In heterokaryons, DNA synthesis is reactivated in macrophage nuclei only in the case of fusion with immortal cells. Assuming that telomerase is responsible for reactivation, the effect of its inhibitor azidothymidine (AZT) was studied in heterokaryons of mouse resident peritoneal macrophages and immortal 3T3 Swiss cells. AZT suppressed reactivation of DNA synthesis in macrophage nuclei and had no effect on DNA synthesis in 3T3 Swiss cell nuclei, suggesting that telomere structure is impaired in normal mouse macrophages.     

Unusual fast sedimenting mitochondria producing heavy DNA in the cells of aging coleoptiles of wheat seedlings

A fraction of unusual fast sedimenting (10 min at 600-1700g) particles with properties of mitochondria has been detected in wheat seedlings. This fraction conventionally called "heavy" mitochondria amounts (by protein) to about 40% of the total subcellular particle fraction sedimented by 10 min centrifugation at 17,000g. The specific feature of these "heavy" mitochondria in aging tissues is an ability to synthesize and even superproduce heavy (rho = 1.718 g/cm3) mitochondrial DNA (H-mtDNA). The share of "heavy" mitochondria sedimented in the interval between 1000 and 1700g and possessing the maximal H-mtDNA synthesis in aging coleoptiles is about 1.5-fold higher than that in young coleoptiles. Although "heavy" mitochondria are present in young plant organs, they seem to be unable to synthesize H-mtDNA; heavy mtDNA forms only in mitochondria of aging or old cells. Thus, aging in plants is accompanied by a change in population of mitochondria and appearance of the ability for selective H-mtDNA superproduction in a certain mitochondrial fraction. Mitochondria isolated from wheat coleoptiles are practically not stimulated by uncouplers. "Heavy" (600-1700g) and usual (4,300-17,400g) mitochondria are similar in respiration rates, cytochrome compositions, cytochrome c amount (per mg protein) and sensitivities to respiration inhibitors. However, "heavy" mitochondria contain (per mg protein) cytochromes b and aa3 by 10-20% and Ca2+ by 2-3-fold more than normal mitochondria. Ultrastructural analysis showed that the isolated fraction of fast sedimenting mitochondria consists of a suspension of closed membrane vesicles filled with cytoplasm and containing one or a few mitochondria. We observed similar structures in situ in vacuoles of parenchyma cells in the apical part of intact coleoptiles. The process of formation of such structures was detected by serial ultra-thin section analysis. It was shown that tonoplast protrudes into vacuoles, the separate mitochondria translocate into these protrusions, and then these structures separate. As a result, the suspended cytoplasmic bodies containing mitochondria appear in vacuoles. Appearance of these bodies containing mitochondria and, in particular, the superproduction of H-mtDNA in them correlate with processes of aging and cell transition to apoptosis.     

Effect of azidothymidine on reactivation of DNA synthesis in macrophage nuclei contained in heterokaryons

EIn heterokaryons, DNA synthesis is reactivated in macrophage nuclei only in the case of fusion with immortal cells. Assuming that telomerase is responsible for reactivation, the effect of its inhibitor azidothymidine (AZT) was studied in heterokaryons of mouse resident peritoneal macrophages and immortal 3T3 Swiss cells. AZT suppressed reactivation of DNA synthesis in macrophage nuclei and had no effect on DNA synthesis in 3T3 Swiss cell nuclei, suggesting an altered telomere structure in normal mouse macrophages.     

Induction of Apoptosis in Rat Myocardium under Anoxic Conditions

The effect of anoxic incubation of small slices of isolated rat hearts on respiration, internucleosomal DNA fragmentation, and mitochondrial ultrastructure was investigated. Anoxic incubation for 72 h induced apoptosis accompanied by internucleosomal DNA fragmentation and changes in respiration and mitochondrial ultrastructure. The mitochondrial population was characterized by morphological heterogeneity. In a significant part of the mitochondrial population there were signs of mitochondrial swelling and appearance of electron-dense mitochondria. Anoxia also induced the appearance of an atypical (and previously unknown) population of small electron-dense mitochondria. They were characterized by unusual localization inside electron-light mitochondria. Under anoxic conditions the inner mitochondrial membrane formed electron-dense ordered structures. All changes described here reflect two opposing processes occurring in mitochondria: apoptotic destruction and compensatory processes responsible for maintenance of mitochondria.     

delta psi-mediated signalling in the bacteriorhodopsin-dependent photoresponse.

It has been shown previously that the proton-pumping activity of bacteriorhodopsin from Halobacterium salinarium can transmit an attractant signal to the bacterial flagella upon an increase in light intensity over a wide range of wavelengths. Here, we studied the effect of blue light on phototactic responses by the mutant strain Pho8l-B4, which lacks both sensory rhodopsins but has the ability to synthesize bacteriorhodopsin. Under conditions in which bacteriorhodopsin was largely accumulated as the M412 bacteriorhodopsin photocycle intermediate, halobacterial cells responded to blue light as a repellent. This response was pronounced when the membrane electric potential level was high in the presence of arginine, active oxygen consumption, or high-background long-wavelength light intensity but was inhibited by an uncoupler of oxidative phosphorylation (carbonyl cyanide 3-chlorophenylhydrazone) and was inverted in a background of low long-wavelength light intensity. The response to changes in the intensity of blue light under high background light was asymmetric, since removal of blue light did not produce an expected suppression of reversals. Addition of ammonium acetate, which is known to reduce the pH gradient changes across the membrane, did not inhibit the repellent effect of blue light, while the discharge of the membrane electric potential by tetraphenylphosphonium ions inhibited this sensory reaction. We conclude that the primary signal from bacteriorhodopsin to the sensory pathway involves changes in membrane potential.     

Role of Telomerase in Reactivation of Macrophage Nuclei in Heterokaryons

It was shown that the duration of stay of macrophages in the peritoneal cavity of mice and method of their isolation did not affect markedly their capacity for resumption of DNA synthesis in heterokaryons. This means that mouse macrophage undergo such changes during differentiation that reactivation of DNA synthesis in their nuclei is only possible after interaction of telomeres with telomerase, since it was already shown that telomerase was involved in reactivation of DNA synthesis in the macrophage nuclei. The results of experiments did not reveal differences in the length of telomeres in mouse macrophages and other somatic cells. This could depend on the significant length of mouse telomeres and, as a result, their shortening, sufficient for the inhibition of proliferation, is beyond the limits of sensitivity of the current methods. It is also possible that changes in DNA properties in the macrophages occurring during their differentiation depend on changes in the conformation of the telomere complex in these cells. Testing of this suggestion is relevant with respect to recent data that cell hybridization, specifically in the form of heterokaryons, may be essential in realization of the therapeutic effect caused by the introduction of cells during cell therapy.