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. The first of these occurs in complex I as a result of the spontaneous release of FMN from the active center. This process is inhibited by adenosine and guanosine phosphates, as well as NAD, but amplified by nicotinamide. The second process is associated with enzymatic hydrolysis of FAD and FMN to riboflavin; it is blocked by EDTA, AMP, NA, NAD. The third process is associated with non-enzymatic hydrolysis of FAD by iron ions in matrix; it is blocked by EDTA and AMP.

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 106 M-1. Specific hydrolysis of rRNA and tRNA by binase during 1-2 min at room temperature leads to a multiple decrease in fluorescence of the dye. This rapid hydrolysis goes to large polynucleotide fragments, but not to short oligonucleotides. The binding constant of binase with rRNA is about of 2.5 × 106 M-1, which is several dozen times higher than with oligonucleotides. The susceptibility to binase attack depends on the secondary structure of RNA, determined by non-canonical ribonucleotides. The developed highly sensitive fluorescent method can be used for the rapid selective detection of trace amounts of rRNA or tRNA, as well as for studying the physicochemical properties of these RNAs. Using the proposed method, one can confidently detect RNA from 10-7 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 1.6 times. Hydrolysis within minutes and even hours led to appearance of large polynucleotide fragments, but not to short oligonucleotides, that was confirmed using polarized fluorescence and highly sensitive measurement of light-scattering. At the moment of the time of formation of a complex between DNA and DNAse I, a strong light-scattering occurred, which then dropped sharply during hydrolysis of high-molecular DNA, and slowly decreased during hydrolysis of fragmented DNA. The proposed methods can be applied for selective detection of trace amounts of various types of DNA, as well as for studying their physic-chemical properties.

[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. It is shown that accumulation of lipofuscin with age occurs in all organs of rats, but least of all in the brain. In addition to fluorescent lipofuscin, non-fluorescent protein aggregates are found in all organs, the largest number of which is found in the heart. Among them, a significant proportion is covalently cross-linked aggregates that are not destroyed by detergents. Their number also increases with age.

[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. However, the content of anthranilic acid, measured by spectrofluorimetry, in adult nematodes is lower than that in the young ones. Artificial aging of nematodes by moderate heating revealed no accumulation of anthranilate and no loss of tryptophan, from which it must be formed. Thus, it is hardly lipofuscin or anthranilic acid. The cause of aging and death of nematodes is the formation of strong cross-links between proteins. This is supported by data on tryptophan fluorescence and light scattering of homogenates: the old worms show a large number of denaturated proteins and large protein particles with a strong cross-links, which are not destroyed be detergent.

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. In the case of 7AAMD with guanine, adenine, thymine and caffeine, the energy is about of 7 kcal/mol, which is a little lower than in the case with DNA (7.7 kcal/mol). On the basis of emission spectra, in all examined compounds, with the exception DNA, the 7AAMD molecule emits photons from water phase, not from a cluster, since photo-excitation leads to desorption of the antibiotic from a cluster surface. We observed also the mutual fluorescence quenching of 7AAMD and FA in their complex. It may well be that this complex forms due to interaction of peptide-lactone rings of 7AAMD with system of FA. In the case of DNA, the complex with 7AAMD has very high stability that is determined not only by interaction between phenoxazine of 7AAMD and the DNA bases, but it is largely owing to the interaction between two peptide-lactone rings of 7AAMD and the DNA deoxyribose-phosphate chains.

[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. A simple method to find true extinction coefficients from the absorption (or excitation) spectra of diluted suspensions (not having multiple light scattering) is suggested. The experimental data on spectra of hemoglobin in erythrocytes, actinomycin in DNA and flavins in mitochondria are given.

[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. A shunt succinate:tetrazolium-reductase activity of protomitochondria was lower and NADH-tetrazolium-reductase activity was higher than that in mitochondria. Electrophoresis and gel filtration found no qualitative differences between protomitochondria, 0.25-0.45 µm in diameter, and mitochondria in major protein composition, but some quantitative differences in several bands were found. Perhaps, these differences reflect the process of intracellular maturation of protomitochondria to mitochondria. The data obtained are important for understanding the mitochondriogenesis in the animal cells.

[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. Thermo-formation of lipofuscin depends on lipid peroxidation, while the presence of lipids is not required for photo-lipofuscin formation. It is shown that the use of detergent able to degrade mitochondria is necessary to measure lipofuscin content properly.

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. Also, we obtained the data that this process is blocked by the enzyme substrate - NADH or by the product - NAD. Recently, we found that this process is strongly blocked by adenine analogs of NAD, contained phosphates: ATP, ADP, and AMP. Adenosine phosphates are able to stabilize the FMN molecule in NADH-dehydrogenase. Using fluorescence spectroscopy and photocolorimetry, we have tested also other natural purine compounds - cAMP, cGMP, GMP, GDP, GTP, IMP, inosine, guanine, and caffeine. It is found that such derivatives of guanine as GMP, GDP, and GTP can prevent the release of FMN into solution. Guanine, cGMP, cAMP and caffeine did not prevent this process. The obtained data allow understand the mechanism of mitochondrial diseases, involving damage of mitochondrial NADH-dehydrogenase, and may help in development of medicines for treatment of these diseases.

[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.

[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. The antibiotic is especially well integrated into the unwound DNA regions. Embedding is accompanied by a long-wavelength shift in the excitation spectrum. The magnitude of the shift was used for calculation of the interaction energy. In the case of AMD with guanine, caffeine and adenine, the value of energy was about of 7 kcal/mol and in the case of fragmented DNA it was only a bit higher: 7.7 kcal/mol. It can be assumed that guanine, adenine, caffeine, and the fragmented DNA could apply as carriers of antibiotic.

[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. The antibiotic is especially well integrated into the unwound DNA regions. Embedding is accompanied by a long-wavelength shift in the absorption spectrum. The magnitude of the shift was used for calculation of the interaction energy. In the case of AMD with caffeine and adenosine, the value of energy is 2.4 and 2.7 kcal/mol and in the case of guanosine and fragmented DNA--considerably higher: 3.3 and 3.7 kcal/mol. It can be assumed that guanosine, adenosine, caffeine, and the fragmented DNA could serve as carriers of antibiotic.

[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.

[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). Safranin O, 8-anilino-1-naphthalene sulfonate and others are poorly linked to the native mitochondria and react only a little to their energization or disconnection. It can be concluded that calculations of the magnitude of the transmembrane potential of the inner mitochondrial membrane in response to the addition of succinate, ATP or dinitrophenol by fluorescence of these probes are incorrect.

[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. The antibiotic effectively interacts with the nucleotides of native DNA at a ratio of 1 : 868, especially strongly with the clusters of satellite fractions and DNA fragments. At low concentrations, it stabilizes the "loose" clusters, i.e., those matching untwisted areas that melt first. At high concentrations, it destabilizes the double helix and causes the aggregation of DNA.

[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. The second method is based on the high-sensitive measurement of light-scattering intensity in aqueous solutions of diluted DHQ preparations using a spectrofluorometer with crossed monochromators. It has been shown that the filtration of DHQ solutions through Millipore filters with a pore diameter of 0.05-0.45 microns makes it possible to nearly completely eliminate long polymers and their aggregates. Long polymers at high concentrations can aggregate. The longest polymers and their aggregates may be 0.1 mm in length, which leads to fluctuations in the light-scattering intensity on the second and minute time scale.

[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.

[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.