Germinal proto-mitochondria from rat liver.

A number of properties of the smallest (less than 0.2 μm) germinal proto-mitochondria (PRMC) from rat liver have been studied. These PRMC were obtained by filtering the light fraction of hepatic mitochondria (MC) through calibrated millipore membranes.

Disruption of flavin homeostasis in isolated rat liver mitochondria.

It has been shown that spontaneous release of non-covalent flavins (from flavoenzymes) begins after isolation of mitochondria from rat liver, which is hydrolyzed to riboflavin. This process is stopped by 1 mM EDTA in the incubation medium. In the presence of NADH, deflavinization of flavoproteins leads to formation of superoxide by at least of three processes.

Fluorescent determination of micro-quantities of RNA using Hoechst 33258 and binase.

Detection of small amounts of RNA in various biological samples is an important applied task. Using fluorescence spectroscopy, the hydrolysis by binase of rRNA and tRNA, stained with Hoechst 33258, in aqueous solutions was investigated. The binding constant of Hoechst with rRNA is 10 M.

Determination of Micro-Quantities of DNA Using DNAse and Fluorescence of Hoechst 33258 and Light-Scattering.

The DNA hydrolysis by deoxyribonuclease (DNAse I) in aqueous solution was studied, using fluorescence spectroscopy and high-sensitive light-scattering detection. Specific hydrolysis of high-polymer DNA or fragmented DNA by the enzyme led to a strong decrease in the fluorescence of the Hoechst dye. The hydrolysis of mitochondrial DNA was accompanied by a decrease in the fluorescence of the dye only in 1.

[Lipofuscin and mitolipofuscin in organs of young and adult rats.].

A comparison of the lipofuscin (aging pigment) content in four organs (liver, kidney, heart, brain) of young and adult rats, as well as in a suspension of hepatic mitochondria, was made. It was demonstrated that mitochondrial lipofuscin - mitolipofuscin, fluorescing in the blue region, makes the main contribution to hepatic lipofuscin. It is assumed that mitolipofuscin in other organs also makes a significant contribution.

[Blue death of nematodes.].

This paper shows that the aging and death of nematodes, accompanied by the ignition of a blue glow under fluorescent microscopy, are not directly linked to any lipofuscin (aging pigment), nor with the anthranilic acid (a product of degradation of tryptophan residues of proteins). The main contribution in the blue flash of the dying nematodes belongs to parasitic light, scattered on the cuticle and bodies of the worm. The main contribution in the blue region at spectrofluorometry of homogenates, obtained from nematodes, really gives anthranilic acid.

[Degradation of Mitochondria to Lipofuscin upon Heating and Illumination].

In the present work, it has been shown that the isolated mitochondria can undergo transformation to lipofuscin granules without any additional factors (oxygen saturation, prooxidants). The process occurs spontaneously and slowly at low temperature, and rapidly--by heating (thermo-lipofuscin) or under UV irradiation (photo-lipofuscin). The main contribution to the formation of mitochondrial lipofuscin comes from denatured proteins.

[Protomitohondria of Liver Cells, Their Similarities and Differences between Mitochondria].

In this paper we continue the study of a number of properties of protomitochondria--small young mitochondrial organelles in the animal cells. Protomitochondria were obtained by filtration of total suspension of mitochondria of rat liver through Millipore filters. Protomitochondria contain an active respiratory chain as evidenced by the high rate of oxygen consumption during succinate and NADH oxidation.

Nucleotide carriers for anti-tumour actinomycin antibiotics.

We have investigated a number of complexes of 7-aminoactinomycin D (7AAMD), with its potential carriers: caffeine, folic acid (FA), purine bases-guanine and adenine, pyrimidine base-thymine and with fragmented DNA to determine more stable and suitable complex. The process of binding of the fluorescent antibiotic with clusters of caffeine, guanine, adenine, thymine and with fragmented DNA was accompanied by a considerable long-wavelength shift in excitation spectrum. The energy of interaction between phenoxazine hetero-cycle of 7AAMD and chromophores of the carriers studied has been found.

[Fluorescence study of energetics in nucleotide-actinomycin complexes].

Variety of different compounds use for delivery of antibiotics to the tumor cells. In this work, using a highly sensitive fluorescence analysis, we have studied complexes of fluorescent analog of the natural heterocyclic antibiotic actinomycin D (7-aminoactinomycin D) with potential carriers: purine bases and fragmented DNA. The antibiotic is not only adsorbed on the surface ofpurine clusters, but also is embedded in them.

[Tyndall's hypochromism in suspensions].

Since the passage of light through each individual particle in a suspension includes the competition of processes of absorption and scattering, it leads to hypochromism--a decrease in the extinction coefficient. Such "scattering" hypochromism increases with the particle size and its refractive index. Since the Tyndall's light scattering in suspensions, where the size of each particle is substantially larger with respect to wavelengths of light, is not strongly dependent on the wavelength, the absorption spectrum (and excitation spectrum) attenuated almost uniformly at different wavelengths.

[Artefacts of confocal microscopy].

Typical artefacts caused by using confocal fluorescent microscopy while studying living cells are considered. The role of light scattering, mobility, staining, local concentrations, etc. is discussed.

[On measurements of mitochondrial transmembrane potential by fluorescent probes].

It is common thought that rhodamine, cyanine, and some other fluorescent dyes are specific potential-dependent and that they allow of quantitatively measuring the transmembrane potential in mitochondria and cells. However, a critical analysis of the experimental data shows that this statement is only a supposition. In reality, widely used fluorescent probes, such as merocyanine 540, Dis-C3-(5).

Stabilization of NADH-dehydrogenase in mitochondria by guanosine phosphates and adenosine phosphates.

It is known that one of the reasons, leading to the development of neuromuscular diseases, including Parkinson's disease, is damage of the mitochondrial NADH-dehydrogenase. Perhaps, it happens when NADH-dehydrogenase loses connection with its coenzyme--flavine mononucleotide (FMN) that occurs at various influences on the enzyme. Previously, we have developed a method, based on fluorescence spectroscopy, to monitor the rate of exit of FMN from isolated mitochondria to solution.

[Energy of interaction in actinomycin-nucleotide complexes].

Variety of different compounds has been used for delivery of antibiotics to the tumor cells. In this work, using the highly sensitive spectrophotometry, the natural complexes of heterocyclic antibiotic actinomycin D (AMD) with such possible curriers like purine and pyrimidine nucleotides as well as fragmented DNA and phospholipid liposomes were studied. The antibiotic is not only adsorbed on the surface of purine clusters, but also is embedded in them.

[Photo-activity of actinomycins].

Using actinomycins as an example, the possibility of increasing the anti-tumor activity of heterocyclic antibiotics due to photo-activation, has been studied. In model experiments with solutions of actinomycins, it was showed that actinomycins have a significant photochemical activity (of its own), changing by the binding to DNA in solution or in tumor cells. Photo-destruction of HeLa cells and the release of the antibiotic were observed.

[Photo-induced conformational motility of proteins].

The dynamics of proteins, detected by fluorescence, consists of three components: spontaneous dynamics, dipole-dipole photo-induced dynamics, thermal photo-induced dynamics. The photo-induced dynamics can lead to activation as well as inactivation of enzymes.

[Cluster melting of DNA-actinomycin complexes].

Sensitive methods of differential UV spectrophotometry and differential scanning microcalorimetry were used to study the interaction of small and large quantities of the natural antitumor antibiotic actinomycin D with clusters of native and fragmented calf thymus DNA during thermal melting. At micromolar (physiological) concentrations, actinomycin is incorporated in untwisted sites of DNA rather than in the double helix. Actinomycin stabilizes these sites and therefore slightly increases the overall melting temperature of DNA.

Melting of DNA-actinomycin clusters.

Differential methods of scanning micro-calorimetry and UV spectrophotometry were used for understanding the interaction of natural anti-tumour antibiotic actinomycin D with cluster sites of native and fragmented DNA during thermal melting. At low (micro-molar) concentrations, the actinomycin molecules penetrate into unwound regions of DNA, but not into the double helix. Moreover, they stabilize the fragmented DNA and increase a total melting point.

Actinomycins like anti-cancer photo-sensitizers.

Spectroscopic and microscopic study on application of actinomycins as anti-tumor photo-sensitizing drugs was carried out in this work. It has been shown that 7-aminoactinomycin (7AAMD) and actinomycin D (AMD) inside cells of line HeLa bind not only with DNA, but also with proteins. Fluorescence of 7AAMD in HeLa cells and destruction of these cells by photosensitizing with actinomycin D were detected.

[Hairpin oligonucleotides as actinomycin carriers].

The redistribution of actinomycin from hairpin oligonucleotide HP1 to dissolved DNA and DNA inside the cell has been discovered and investigated. It was found that the penetration of the antibiotic in a complex with HP1 into cancer cells takes place more effectively than that of the antibiotic separately. It is suggested that hairpin oligonucleotides can serve as molecular carriers of heterocyclic antibiotics to cancer cells.

[Detection of polymeric forms of dihydroquercetin by optical absorption and light scattering].

Two methods for the detection of long polymers in dihydroquercetin (DHQ) preparations has been developed. The first method is based on UV spectrophotometry. It was shown that the quantity of long polymers in aqueous solutions can be estimated by the ratio of the absorption bands at 328 and 290 nm, since the 328-nm band was attributed to the monomeric form of DHQ, whereas the 290-nm band was attributed to both the monomeric and polymeric forms.

[7-aminoactinomycin as a fluorescent probe for DNA unwinding and denaturation].

A fluorescent analogue of the antibiotic actinomycin D, 7-aminoactinomycin D (7AAMD), which is widely used in molecular biology, was shown by steady-state, polarization, and phase fluorescent spectroscopy to bind primarily in unwound regions of DNA with a concomitant increase in its emission intensity. The maximum emission intensity of 7AAMD is observed for denatured DNA. Thus, 7AAMD may serve as a good indicator of DNA unwinding, denaturation, and fragmentation.

Effect of Ca2+ gradient on the structure of sarcoplasmic reticulum membranes.

The structure of sarcoplasmic reticulum membranes was studied in the presence of modeled transmembrane Ca2+ gradient corresponding to the status of Ca2+ depot at different stages of the muscle contraction-relaxation cycle in health and disease. Various sites of the membrane were characterized using spectral analysis of tryptophan, pyrene, and merocyanine-540 fluorescence without evaluating specific changes in the molecules of membrane components (Ca2+ -ATPase, ryanodine receptor, and lipids). The transmembrane Ca2+ gradient modulates the protein-lipid interactions and structural characteristics of the membrane.

[Caffeine clusters as transmitters of actinomycin antibiotics to DNA in solutions].

Using the screening model of hypochromism, we showed that caffeine forms regular clusters consisting of 8-12 molecules. Addition of 7-aminoactinomycin D (7AAMD, a fluorescent analogue of actinomycin D) to the clusters leads to its sorption on the cluster surface. Photoexcitation of 7AAMD leads to its desorption from the surface into the aqueous phase and emission of a quantum.