Interaction of cyanine dyes with nucleic acids: XXXI. Using of polymethine cyanine dyes for the visualization of DNA in agarose gels

Fifteen polymethine cyanine dyes were studied as fluorescent stains for DNA in electrophoretic gels. Among studied cyanines, two dyes CPent V and CCyan 2-O most effectively visualized covalently closed and linear double-stranded DNA molecules in gels under standard conditions using UV-illumination, green filter and black-and-white photo film. Ethidium bromide was 1.2-1.6 times more effective as compared to cyanine dyes in staining of DNA in the concentration range of 8-18 ng, while studied cyanines were more sensitive to DNA quantity above 50 ng.     

Dynamics of characteristics of electrical activity recorded from tropho-and ergotropic zones of the rat hypothalamus in long-term emotional stress

We examined the dynamics of the spectral powers and indices of frequency components of background field electrical activity recorded from tropho-and ergotropic zones of the rat hypothalamus (electrohypothalamogram, EHtG) in the course of long-term (21 weeks) emotional stress induced by a zooconflict situation. Low-frequency delta-range (0.5 to 3.5 Hz) oscillations dominated in EHtGs recorded from both hypothalamic zones of both control and stressed animals. The integral power of EHtG oscillations underwent significant changes within the observation period; it considerably increased on the 6th to 9th week, dropped within the 12th to 18th week, and, after this (up to the 21st week) again increased, i.e., the dynamics, in general, were threephase. The dynamics of the power of separate frequency components of EHtG showed certain similarities to the dynamics of the integral power. Changes in the EHtG power in stressed animals were characterized by a shorter duration and greater rate; the powers of all EHtG components in these animals were lower than in control ones within a greater part of the observation period. The dynamics of the normalized powers and of the indices of different EHtG rhythms in stressed and control animals, especially the dynamics of dominating oscillations of the delta and theta ranges, were to a certain extent opposite. Our data show that changes in the EHtG can be electrographic correlates of subsequent phases of a long-lasting stress reaction of the organism in the course of the experiment; specific features of manifestation of these modifications in the tropho-and ergotropic hypothalamic zones are related to a certain specificity of the hormonal/transmitter mechanisms in the structures under study. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 69–80, January–February, 2007.     

Dynamics of the Indices of Electrohippocampogram Recorded from Rats under Conditions of Long-Lasting Stress: Effects of Modulators of Synaptic Transmission

We studied the effects of modulation of the activity of noradrenergic and GABA-ergic cerebral transmitter systems on the hippocampal electrical activity in rats during long-lasting (21 weeks) stress induced by a zooconflict situation. The background field electrical activity of the hippocampus (electrohippocampogram, EHcG) was recorded from control and stressed rats in the state of residual ketamine-barbiturate anesthesia. We used injections of the following modulators of central neurotransmission: amitriptyline, aminazine, and carbamazepine, which are extensively used in clinics for intensification of the activity of the antistress GABA system based on the influence of these agents on the monoaminergic cerebral systems. Under such conditions, we found the two-phase dynamics of spectral powers of EHcG components. In the first phase, where changes in the excitatory influences on central neurons prevailed, the total power of EHcG oscillations and spectral powers of its components were significantly lower with respect to the control, while in the second phase we observed rapid increases in the above indices, which probably resulted from parallel hyperactivation of both excitatory and inhibitory (GABA-ergic) elements of neuronal networks of the hippocampus. In rats forming the groups for comparison (control animals and stressed ones with no injections of synaptic modulators), changes in the powers of EHcG oscillations during the 21-week-long experiment waves were three-phase; in the latter animal group, the above characteristics differed from each other mostly quantitatively. Such changes in stressed rats with respect to the control ones could reflect modulation of the mediator/hormonal influences on hippocampal neurons during three phases of the stress reaction of the organism.     

Evolution of the human immunodeficiency virus type 1 subtype-specific V3 domain is confined to a sequence space with a fixed distance to the subtype consensus.

Human immunodeficiency virus type 1 (HIV-1) strains can be separated into genetic subtypes based on phylogenetic analysis of the envelope gene. Once it had been shown that population-wide intrasubtype genetic variation of HIV-1 strains increases in the course of the AIDS epidemic, it remained uncertain whether HIV-1 subtypes are phenotypic entities spreading as distinct virus populations. To examine this, we applied Eigen's concepts of sequence geometry and fitness topography to the analysis of intrasubtype evolution of the gp120 V3 domain of HIV-1 subtypes A, B, C, and D in the course of the global AIDS epidemic. We observed that despite the high evolution rate of HIV-1, the nonsynonymous distances to the subtype consensus of sequences obtained early in the epidemic are similar to those obtained more than 10 years later, in contrast to the synonymous distances, which increased steadily over time. For HIV-1 subtype B, we observed that the evolution rate of the individual sequences is independent of their distance from the subtype B consensus, but for the individual sequences most distant from the consensus evolution away from the consensus is constrained. As a result, individual HIV-1 genomes fluctuate within a sequence space with fixed distance to the subtype consensus. Our findings suggest that the evolution of the V3 domain of HIV-1 subtypes A, B, C, and D is confined to an area in sequence space within a fixed distance to the consensus of a respective subtype. This in turn indicates that each HIV-1 subtype is a distinct viral quasispecies that is well adapted to the present environment, able to maintain its identity in the V3 region over time, and unlikely to merge during progression of the AIDS epidemic.     

Prediction of the three-dimensional structure of aflatoxin of Aspergillus flavus by homology modelling

Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus spp. The toxic effects of aflatoxins have adverse consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a protein containing a zinc-finger DNA-binding motif. AFLR-Protein three-dimensional model was generated using Robetta server. The modeled AFLR-Protein was further optimization and validation using Rampage. In the simulations, we monitored the backbone atoms and the C-α-helix of the modeled protein. The low RMSD and the simulation time indicate that, as expected, the 3D structural model of AFLR-protein represents a stable folding conformation. This study paves the way for generating computer molecular models for proteins whose crystal structures are not available and which would aid in detailed molecular mechanism of inhibition of aflatoxin.     

The molecular epidemiological characteristics of the HIV-1 variants circulating among injection drug addicts on the territory of the CIS Nations

Different strains of HIV-1, circulating among drug addicts introducing drugs intravenously and detected on the territories of Russia, Ukraine and Belarus, have been characterized by the methods of the comparative analysis of genetic sequences of different variants of HIV (gene typing) and the study of the immunological properties of autoimmune sera (serotyping).     

Shortening of the Symptom-Free Period in Rhesus Macaques Is Associated with Decreasing Nonsynonymous Variation in the env Variable Regions of Simian Immunodeficiency Virus SIVsm during Passage

During six blood passages of simian immunodeficiency virus SIVsm in rhesus macaques, the asymptomatic period shortened from 18 months to 1 month. To study SIVsm envelope gene (env) evolution during passage in rhesus macaques, the C1 to CD4 binding regions of multiple clones were sequenced at seroconversion and again at death. The env variation found during adaptation was almost completely confined to the variable regions. Intrasample sequence variation among clones at seroconversion was lower than the variation among clones at death. Intrasample variation among clones from a single time point as well as intersample variation decreased during the passage. In the variable regions, the mean number of intrasample nonsynonymous nucleotide substitutions decreased from the first passage (5.26 × 10⁻² ± 0.6 × 10⁻² per site) to the fifth passage (2.24 × 10⁻² ± 0.4 × 10⁻² per site), whereas in the constant regions, the mean number of intrasample nonsynonymous nucleotide substitutions differed less between the first and fifth passages (1.14 × 10⁻² ± 0.27 × 10⁻² and 0.80 × 10⁻² ± 0.24 × 10⁻² per site). Shortening of the asymptomatic period coincided with a rise in the Ks/Ka ratio (ratio between the number of synonymous [Ks] and the number of nonsynonymous [Ka] substitutions) from 1.080 in passage one to 1.428 in passage five and mimicked the difference seen in the intrahost evolution between asymptomatic and fast-progressing individuals infected with human immunodeficiency virus type 1. The distribution of nonsynonymous substitutions was biphasic, with most of the adaptation of env variable regions occurring in the first three passages. This phase, in which the symptom-free period fell to 4 months, was followed by a plateau phase of apparently reduced adaptation. Analysis of codon usage revealed decreased codon redundancy in the variable regions. Overall, the results suggested a biphasic pattern of adaptation and evolution, with extremely rapid selection in the first three passages followed by an equilibrium or stabilization of the variation between env clones at different time points in passages four to six.     

Interaction of cyanine dyes with nucleic acids. XXI. Arguments for half-intercalation model of interaction

The spectral luminescent properties of two groups of monomethine cyanine dyes were studied in the presence of DNA. The first group included five dyes with 5,6-methylenedioxy-[d]-benzo-1,3-thiazole heterocycle and their unsubstituted analogs. Five monomethine pyrylium cyanines and their N-methyl-pyridine analogs were included in the second group. In each pair the pyrylium and pyridine dyes had similar geometry but differed in charge density distribution. The results presented some evidence in favor of the half-intercalation interaction mode between the studied dyes and DNA. When the benzothiazole residue had the lowest electron donor ability between the two heterocycles in the dye molecule, its substitution with the bulky methylenedioxy group led to a significant decrease in fluorescence enhancement of the dye-DNA complex. On the contrary, when the substituents that create steric hindrance (e.g., methylenedioxy and methyl groups) were introduced into the heterocycle with the higher electron donor ability, the fluorescence enhancement value of the dye-DNA complex was virtually unchanged. The changes in the Stock's shift values upon the formation of the dye-DNA complexes were in agreement with the proposed half-intercalation model. Interestingly, in the dye-DNA complexes the pyrylium dyes probably resided in a place similar to the pyridine ones. It is possible that the benzothiazole (or benzooxazole) ring intercalated between the DNA bases and the pyrylium (or pyridine) residue was located in the DNA groove closer to the phosphate backbone.