Changes in localization of cellular vesicular apparatus during differentiation of myoblasts into myotubules in cell culture

A study of changes in intracellular processes in the course of the differentiation of myoblasts into myotubules is of great importance for understanding fundamental problems of cell biology. This primarily concerns the change in the spatial organization of the vacuolar apparatus, which reflects changes in the properties of the membranes, cytoskeleton, and processes of vesicular transport in the course of differentiation. In the present work, the distribution of acid organelles (lysosomes, late endosomes, Golgi cisternae) accumulating acridine orange during the process of the formation of myotubules in the L6J1 cell culture is analyzed. The perinuclear localization of acid cell organelles in myoblasts is shown to be replaced by the diffuse distribution of these organelles throughout the entire volume of the myotubule cytoplasm. The use of lipophilic dyes RH 414 or di-8-ANEPPS enables one to compare the process of formation and dynamics of endocytic vesicles in myoblasts and myotubules. For the analysis of nonspecific endocytosis, we also used the semiconductor nanocrystals, i.e., quantum dots (QDs) conjugated with TAT-peptide, a cell-penetrating peptide. We have shown that the QD-TAT complexes enter into myoblasts via endocytosis. It has been established that QD-TAT complexes penetrate myoblasts, but do not penetrate myotubules, even after incubation for 24 h, which can indicate changes in the properties of the plasma membrane in the process of differentiation of skeletal muscle fibers.     

Pulse-electrotypes of Listeria monocytogenes strains, isolated in Moscow

The characterization of the pulse-electrotypes of L. monocytogenes, isolated in 2003-2004 in Moscow from different sources, is presented. Among the cultures, isolated from humans, one outbreak pulse electrotype was detected and from different objects in buildings where a wide variety of food products was produced several probably related and unrelated pulse-electrotypes were obtained. The conclusion was made that several independent L. monocytogenes clones existed on the territory of Moscow, and many products supplied to retail trade and public catering enterprises were contaminated with these clones. Pulse electrophoresis was shown to be the most effective method for intraspecific typing and the study of the molecular epidemiology of listeriosis. Grounds for the necessity to improve the microbiological diagnostics of L. monocytogenes infection are given.     

Characterization of Escherichia coli strains O157:H7, isolated on the territories of the Central Federal District

The characterization of E.coli strains O157:H7, isolated from humans and animals on some territories of the Central Federal District, is presented. Among the isolates from human outbreaks, related and, probably, related cultures prevailed, while among the isolates obtained from different animals mainly unrelated cultures have been detected. A conclusion has been made concerning the existence of several independent zoonotic reservoirs of E. coli O157:H7 infection on this territory. The advantages and drawbacks of the use of pulse electrophoresis in the characterization of E. coli O157:H7 are discussed. Grounds are given for the necessity of the patients examination with hemorrhagic enetrocolitis for the presence of E. coli O157:H7, as well as for the expediency of having a special item for the registration of this E. coli infection in relevant statistical forms.     

Acridine orange distribution and fluorescence spectra in myoblasts and single muscle fibers

Acridine orange (AO) fluorescence spectra in nuclei and cytoplasm of living myoblasts L6J1 and frog single muscle fibers have been studied using spectral scanning system of Leica TCS SL confocal microscope. AO fluorescence spectra in salt solutions dependent on free AO concentrations or in complex with DNA have also been obtained. Myoblast nuclei fluoresced in the green spectral region with maximum at about 530 nm; nucleoli had the brightest fluorescence. The fluorescence of nuclear chromatin was not uniform. Similar fluorescence of nuclei and nucleoli was observed in frog single muscle fibers. Uniform, weak, green fluorescence was observed in the myoblast cytoplasm. In the sarcoplasm of muscle fibers, AO green fluorescence was seen in A discs. In the cytoplasm of myoblasts and muscle fibers stained with AO, different red, yellow, and green fluorescent granules, which were acidic organelles, were visualized. The comparison of AO fluorescence spectra in living cells with AO fluorescence spectra in buffer solutions with different AO concentrations and AO in complex with DNA enables the estimation of the AO concentration in acidic granules. It is important for the evaluation of these cellular organelles functions in intracellular transport, adaptation, and apoptosis, as well as in a number of pathological processes.     

Natural foci of leptospirosis on the territory of Moscow in 1995 - 2004

The results of ten-year observations of the natural foci of leptospirosis on the territory of Moscow are presented. Information on the foci, the main species of small mammals (the reservours of the infection), the etiological structure of leptospires, circulating among rodents and insectivores, is given.     

Untargeted quantum dots in confocal microscopy of living cells

The problem of the nonspecific binding of quantum dots (QDs) with cells is very important, but not fully understood taking into account the possible application of QDs in medical and fundamental studies. The interactions of untargeted CdSe/ZnS QDs with isolated frog muscle fibers, HeLa cells, and J774 cells were investigated. The observations were made on living cells using laser confocal microscopy (Leica TCS SL). QDs covered with polyethylene glycol without any functional reactive groups with an emission maximum at 565 nm were used in the study. This type of QD is suggested to prevent the interaction of QDs with biological molecules. It has been shown that QDs do not enter HeLa cells, the T-system, or the sarcoplasm of skeletal muscle fibers. However, during long-term incubation, J774 cells can take up QDs. The obtained data demonstrated the diversity of interactions of untargeted QDs with different cell types and are important for understanding problems of nonselective uptake and cytotoxicity of QDs.     

The functional morphology of polytene chromosomes in the midge Chironomus pilicornis F. from the permafrost zone reservoirs

The karyotype and chromosomal polymorphism of Chironomus pilicornis from the reservoirs of Yakutian permafrost zone are described. In the Yakutian populations, of 10 inversion sequences 11 genotypical combinations of these were registered. The level of inversion heterozygosity is 50-70%, but in several populations it makes only 11-29%. In comparison with the Scandinavian populations, significant functional modifications of chromosomal morphology were found. They are associated with the increase in centromeric heterochromatin amount, facultative chromocentre formation, and the appearance of numerous B-chromosomes and nucleoli, including the facultative ones. A possible adaptive significance of such functional modifications conditioned by climate peculiarities is discussed.     

Quantum Dots based on Indium Phosphide (InP): the Effect of Chemical Modifications of the Organic Shell on Interaction with Cultured Cells of Various Origins

CdSe and CdTe-based semiconductor fluorescent nanocrystals, also called quantum dots (QDs), attract the attention of biologists due to their wide range of emission in a visible light interval, high fluorescence quantum yield and photostability. However, their application is limited because of possible toxicity of cadmium. Indeed, there is a probability of metal leakage from QDs cores as a result of damage of both inorganic and organic layers of shells covering QDs. An alternative to cadmium QDs could be nanostructures having as a core, for example, non-toxical indium phosphide (InP), also emitting in the visible region of the spectrum. At present, there is few works on the use of these particles in biology. In this study, a comparative analysis of the spectral-luminescent properties of two InP/ZnS-QDs samples coated with PEG carrying- COOH or -NH₂ functional groups was performed. The obtained data were compared with the characteristics of CdSe/ZnS-QDs coated with PEG. The photophysical properties of all QDs in aqueous solution corresponded to the information claimed by manufacturers, but the fluorescence quantum yield of InP-based nanoparticles was found to be lower than that of CdSe-QDs. We also show that the photoluminescence of all types of QDs at pH 4.0 was lower than at pH 7.4, while the decrease in fluorescence intensity was minimal in the case of QDs-PEG-COOH. Studying the uptake of all three types of QDs by J774 macrophages, we found that the fluorescence spectra of internalized QDs do not change in comparison with those in solution. All three types of QDs after 24 hours of incubation were accumulated in the cells, but while QDs-NH₂ and QDs without reactive groups were detected mainly in vesicular-like discrete structures, the QDs-COOH were diffusely distributed throughout the cytoplasm. This fact indicates different mechanisms of interaction with cell membranes. In nonphagocytic HeLa cells all types of QDs behaved similarly, but the overall level of cells fluorescence was much lower. This may be due to both reduced nonspecific uptake and possible quenching of QDs fluorescence in acidic endolysosomes. Cytofluorimetric analysis of propidium iodide accumulation showed that after 24 hours incubation with all studied types of QDs as well as in control (no QDs), the proportion of dead HeLa cells did not exceed 10%. Thus, it has been demonstrated that non-toxic InP-based QDs can be used as an effective tool for biological research.     

Rodents as the vectors of infective agents in the natural foci of infections in the Moscow megapolis

This work deals with the results of 40-year observations on the circulation of infective agents in the natural foci of infections (tularemia, leptospirosis, hemorrhagic fever with renal syndrome) among rodents and other small mammals in the territory of the Moscow. The monitoring of their frequency and the infection rates remains the main effective measure for the prophylaxis of dangerous infections among the population of the megapolis.