Peptidase activity of the fetoplacental complex in the norm and pathology

The activity of angiotensin converting enzyme (ACE), carboxypeptidase N (CPN), and leucine aminopeptidase (LAP) has been investigated in the fetoplacental complex (FPC) in norm and fetoplacental insufficiency (FPI). ACE and LAP activities were significantly higher in the placental tissue than in maternal serum and umbilical vein serum. CPN activity was significantly lower in umbilical vein serum as compared to that of women in childbirth. Probably, the studied enzymes are involved in formation of reduced vascular sensitivity of FPC during physiological pregnancy. In cases of placental insufficiency a significant increase of LAP activity was found in the placental tissue and umbilical vein serum. In addition, the pathological course of pregnancy caused a significant increase of CPN activity in serum of pregnant women in comparison to the norm. The obtained data suggest that during FPI proteolytic enzymes participate in the formation of compensatory-adaptive reactions in the FPC. Results of this study are interesting in the context of development of methods for prevention and correction of metabolic disorders in pathologies of pregnancy.     

Modeling of mechanoelectric coupling in cardiomyocytes in norm and pathology

We developed mathematical models of the electromechanical function of cardiomyocytes and the simplest mechanically heterogeneous myocardial systems, muscle duplexes. By means of these models we studied the contribution of mechanoelectric feedbacks to the contractile activity of the myocardium in norm and pathology. In particular, we simulated and clarified the effects of mechanical conditions on both the form and the duration of the action potential during contractions. From this standpoint different kinds of myocardium mechanical heterogeneity were analyzed. As we have established, the latter can play both a positive and a negative role, depending on the distribution of mechanical nonuniformity and the sequence of activation of heterogeneous myocardium system elements. By means of the same models, we studied the contribution of mechanical factors to the arrhythmogenicity in the case of the cardiomyocyte calcium overload caused by the attenuation of the sodium-potassium pump and outlined the ways for correcting the contractile function in these disturbances.     

Shaping processes in drying drops of serum in norm and pathology

The results of physical and mathematical simulation of the drying process for colloid liquids under different initial conditions were considered. It was shown that the concentration and stability of a colloid solution play an important role in the shaping of drying drops. Possible mechanisms of the formation of morphological differences in dried drops of serum in healthy and ill people are discussed.     

Structure and function of enteric alpha defensins in norm and pathology

This review summarizes currently available data on enteric alpha defensins structure, their functions in the innate and adaptive immunity systems and the role in development of intestinal illnesses.     

Anthracycline-DNA interactions studied with linear dichroism and fluorescence spectroscopy

DNA-binding geometry and dynamics of a number of anthracyclines, including adriamycin and 4-demethoxydaunorubicin, interacting with DNA have been studied by means of linear dichroism and fluorescence techniques. The anthracycline chromophore is found to be approximately parallel to the plane of the DNA bases and to have a restricted mobility, as would be expected for an intercalative binding mode, but there are variations between different directions in the chromophore as well as between the drugs. From dichroic spectra of adriamycin in an anisotropic host of poly(vinyl alcohol), absorption components corresponding to transitions with mutually orthogonal polarizations have been resolved. These can be exploited to determine the orientations of the two chromophore axes in the DNA complex relative to the DNA helix axis. In a certain binding regime the long axis of the bound anthracycline chromophores (with the exception of 4-demethoxydaunorubicin) is found to be approximately 10 degrees closer to perpendicular to the helix axis than are the DNA bases. This demonstrates that the average base tilt is at least 10 degrees. By contrast, the short axis of the aglycon moiety is found to be tilted some 20-30 degrees from perpendicular. This may be because it is probing a base direction with a more pronounced, static or dynamic, inclination than the average in DNA. The drug orientation and the DNA orientation (reflecting flexibility) are observed to vary differently and nonmonotonically with binding ratio, suggesting specific binding and varying site geometries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of interleukin-1 beta mRNA expression in the norm and pathology]

We investigated cytoplasmic RNA from the peripheral blood cells of pollinosis patients. Increased levels of IL-I mRNA were registered in neutrophils of patients suffering from pollinosis for a long time. Neutrophils from patients at the time of exacerbation of pollinosis responded to stimuli less than the cells from healthy donors. During remission the activated neutrophils from pollinosis patients and those from healthy donors had same levels of IL-I mRNA.     

Processive cytoskeletal motors studied with single-molecule fluorescence techniques

Processive cytoskeletal motors from the myosin, kinesin, and dynein families walk on actin filaments and microtubules to drive cellular transport and organization in eukaryotic cells. These remarkable molecular machines are able to take hundreds of successive steps at speeds of up to several microns per second, allowing them to effectively move vesicles and organelles throughout the cytoplasm. Here, we focus on single-molecule fluorescence techniques and discuss their wide-ranging applications to the field of cytoskeletal motor research. We cover both traditional fluorescence and sub-diffraction imaging of motors, providing examples of how fluorescence data can be used to measure biophysical parameters of motors such as coordination, stepping mechanism, gating, and processivity. We also outline some remaining challenges in the field and suggest future directions.     

Human serum albumin interaction with formononetin studied using fluorescence anisotropy, FT-IR spectroscopy, and molecular modeling methods

Interaction of formononetin with a model transport protein, human serum albumin (HSA), has been studied using fluorescence anisotropy, FT-IR spectroscopy, and molecular modeling methods. Upon binding with HSA, the fluorescence spectrum of formononetin exhibits appreciable hypsochromic shift along with an enhancement in the fluorescence intensity. Gradual addition of HSA led to a marked increase in fluorescence anisotropy (r). From the value of fluorescence anisotropy, it is argued that the drug is located in a restricted environment of protein. The binding constant (K approximately 1.6 x 10(5) M(-1)) and the standard free energy change (DeltaG(0) approximately -29.9 kJ/mol) of formononetin-HSA interaction have been calculated according to the relevant fluorescence data. Fourier transform infrared measurements have shown that the secondary structures of the protein have been changed by the interaction of formononetin with HSA. Computational mapping of the possible binding sites of formononetin revealed the molecule to be bound in the large hydrophobic cavity of subdomain IIA.     

RNA conformation and folding studied with fluorescence resonance energy transfer

Fluorescence resonance energy transfer (FRET) results from nonradiative coupling of two fluorophores and reports on distances in the range 10-100 A. It is therefore a suitable probe to determine distances in RNA molecules and define their global structure, to follow kinetics of RNA conformational changes during folding in real time, to monitor ion binding, or to analyze conformational equilibria and assess the thermodynamic stability of tertiary structure conformers. Along with the basic principles of steady-state and time-resolved fluorescence resonance energy transfer measurements, approaches to investigate RNA conformational transitions and folding are described and illustrated with selected examples. The versatility of FRET-based techniques has recently been demonstrated by implementations of FRET in high-throughput screening of potential drugs as well as studies of energy transfer that monitor RNA conformational changes on the single-molecule level.     

Human pathology in NCL

In childhood the neuronal ceroid lipofuscinoses (NCL) are the most frequent lysosomal diseases and the most frequent neurodegenerative diseases but, in adulthood, they represent a small fraction among the neurodegenerative diseases. Their morphology is marked by: (i) loss of neurons, foremost in the cerebral and cerebellar cortices resulting in cerebral and cerebellar atrophy; (ii) an almost ubiquitous accumulation of lipopigments in nerve cells, but also in extracerebral tissues. Loss of cortical neurons is selective, indiscriminate depletion in early childhood forms occurring only at an advanced stage, whereas loss of neurons in subcortical grey-matter regions has not been quantitatively documented. Among the fourteen different forms of NCL described to date, CLN1 and CLN10 are marked by granular lipopigments, CLN2 by curvilinear profiles (CVPs), CLN3 by fingerprint profiles (FPPs), and other forms by a combination of these features. Among extracerebral tissues, lymphocytes, skin, rectum, skeletal muscle and, occasionally, conjunctiva are possible guiding targets for diagnostic identification, the precise type of NCL then requiring molecular analysis within the clinical and morphological context. Autosomal-recessive adult NCL has been linked molecularly to different childhood forms, i.e. CLN1, CLN5, and CLN6, whilst autosomal-dominant adult NCL, now designated as CLN4, is caused by a newly identified separate gene, DNAJC5. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.     

Correspondence between spectral power and synchronization of the brain rhythms in the norm and cognitive pathology

The work is aimed at the study of correlations between the measures of spectral power and cortical interactions of EEG rhythms in healthy subjects and schizophrenic patients ("acute" and chronic cases). All brain rhythms in healthy subjects appeared to be symmetrical and synchronous both in phase and frequency. In "acute" schizophrenics, opposite to healthy subjects, the distribution of cortical activity is asymmetrical, and in the chronic cases, the spectral power of most cortical rhythms is decreased as compared to healthy subjects. In the "acute" patients, interhemispheric connections are absent in all rhythms but alpha. In the chronic patients, the number of cortical connections is slightly higher than in the acute patients; and they are located in the posterior areas in the gamma rhythm. These neurophysiological aberrations evidently underlie the multiple mental activity disorders in schizophrenic patients. Thus, the correspondence between the brain rhythms and their synchronization is a necessary condition for normal perception, emotions and cognition evidently influencing behavior and consciousness.     

Polyamines and collagenes interaction in different processes of extracellular matrix production and functioning in norm and with pathology

A great file of experimental data on tight interrelations between polyamines and main components of extracellular matrix (collagenes, laminin, fibronectin, etc.) metabolisms has been accumulated in the recent twenty years. Consequences of such tight interaction of collagenes, polyamines, enzymes of their metabolisms during formation and functioning of extracellular matrix are realised not only at a cellular level (proliferation, differentiation, adhesion, apoptosis), but also at the level of the whole organism (immunity, connective tissues and skeleton formation, tumors). Search for the ways of cells behavior modulation via influence on the levels of polyamines and contents of extracellular matrix at present is new direction in pharmacology of antitumor preparations that enables regulation of organism-tumor relationships, inhibition of angiogenesis, tumor growth and metastasing.     

State transitions in cyanobacteria studied with picosecond fluorescence at room temperature

Cyanobacteria can rapidly regulate the relative activity of their photosynthetic complexes photosystem I and II (PSI and PSII) in response to changes in the illumination conditions. This process is known as state transitions. If PSI is preferentially excited, they go to state I whereas state II is induced either after preferential excitation of PSII or after dark adaptation. Different underlying mechanisms have been proposed in literature, in particular i) reversible shuttling of the external antenna complexes, the phycobilisomes, between PSI and PSII, ii) reversible spillover of excitation energy from PSII to PSI, iii) a combination of both and, iv) increased excited-state quenching of the PSII core in state II. Here we investigated wild-type and mutant strains of Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 using time-resolved fluorescence spectroscopy at room temperature. Our observations support model iv, meaning that increased excited-state quenching of the PSII core occurs in state II thereby balancing the photochemistry of photosystems I and II.     

Membrane mobility and microdomain association of the dopamine transporter studied with fluorescence correlation spectroscopy and fluorescence recovery after photobleaching

To investigate microdomain association of the dopamine transporter (DAT), we employed FCS (fluorescence correlation spectroscopy) and FRAP (fluorescence recovery after photobleaching). In non-neuronal cells (HEK293), FCS measurements revealed for the YFP-DAT (DAT tagged with yellow fluorescent protein) a diffusion coefficient (D) of approximately 3.6 x 10(-9) cm2/s, consistent with a relatively freely diffusible protein. In neuronally derived cells (N2a), we were unable to perform FCS measurements on plasma membrane-associated protein due to photobleaching, suggesting partial immobilization. This was supported by FRAP measurements that revealed a lower D and a mobile fraction of the YFP-DAT in N2a cells compared to HEK293 cells. Comparison with the EGFP-EGFR (epidermal growth factor receptor) and the EGFP-beta2AR (beta2 adrenergic receptor) demonstrated that this observation was DAT specific. Both the cytoskeleton-disrupting agent cytochalasin D and the cholesterol-depleting agent methyl-beta-cyclodextrin (mbetaCD) increased the lateral mobility of the YFP-DAT but not that of the EGFP-EGFR. The DAT associated in part with membrane raft markers both in the N2a cells and in rat striatal synaptosomes as assessed by sucrose density gradient centrifugation. Raft association was further confirmed in the N2a cells by cholera toxin B patching. It was, moreover, observed that cholesterol depletion, and thereby membrane raft disruption, decreased both the Vmax and KM values for [3H]dopamine uptake without altering DAT surface expression. In summary, we propose that association of the DAT with lipid microdomains in the plasma membrane and/or the cytoskeleton serves to regulate both the lateral mobility of the transporter and its transport capacity.     

Intracerebral EEG functioning as a reflexion of the systemic brain organization in norm and pathology

The authors summarized the EEG findings and defined the nature of intercentral EEG relationships in different functional states of healthy subjects and patients with organic cerebral pathology based on coherence analysis. The EEG features typical of healthy subjects were identified: an anterior-posterior gradient of the mean coherence and the character of cortical-subcortical relationships in the anterior cerebral structures. Right- and lefthanded subjects showed the frequency and regional differences in EEG coherence, which reflected, mainly, specific intracortical relationships. Development and regression of pathologic signs in right- and lefthanded patients with organic brain lesions are thought to be determined by these differences. As distinct from cortical pathology, lesions of regulatory structures (diencephalic, brainstem, and limbic) were shown to produce more diffuse changes in intercentral relationships with a tendency to reciprocity. Intercentral relations, including their interhemispheric differences, varied with changes in the functional state of healthy subjects (increase and decrease in the level of functioning). A certain time course of changes in intercentral relationships was also revealed in patients with organic brain lesions during recovery of their consciousness and mental activity. Changes in the dominance of activity of individual regulatory structures are considered to be one of the most important factors that determine the dynamic character of EEG coherence.     

Mechanism of the efficient tryptophan fluorescence quenching in human gammaD-crystallin studied by time-resolved fluorescence

Human gammaD-crystallin (HgammaD-Crys) is a two-domain, beta-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HgammaD-Crys has four highly conserved buried tryptophans (Trps), with two in each of the homologous beta-sheet domains. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. The dispersal of the excited-state energy to avoid covalent damage is likely to be physiologically relevant for the lens crystallins. Trp fluorescence is efficiently quenched in native HgammaD-Crys. Previous steady-state fluorescence measurements provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain [Chen, J., et al. (2006) Biochemistry 45, 11552-11563]. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron transfer to the amide backbone. Here we report additional information obtained using time-resolved fluorescence spectroscopy. In the single-Trp-containing proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly quenched Trp68 and Trp156 have very short lifetimes, tau approximately 0.1 ns, whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, tau approximately 3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trp130) decreased from approximately 3 to approximately 1 ns. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. The absence of a time-dependent red shift in the time-resolved emission spectra of Trp130 proves that its local environment is very rigid. Time-resolved fluorescence anisotropy measurements with the single-Trp-containing proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect HgammaD-Crys from excited-state reactions causing permanent covalent damage.     

DNA tetraplex formation studied with fluorescence resonance energy transfer.

It is emerging that DNA tetraplexes are pivotal for many major cellular processes, and techniques that assess their structure and nature to the point are under development. Here we show how the structural conversion of largely unstructured single-stranded DNA molecules into compact intrastrand DNA tetraplexes can be monitored by fluorescence resonance energy transfer. We recently reported that intrastrand tetraplex formation takes place in a nuclease hypersensitive element upstream of the human c-myc proto-oncogene. Despite the highly repetitive guanine-rich sequence of the hypersensitive element, fluorescence resonance energy transfer measurements indicate that only one well defined tetraplex structure forms therein. The proposed structure, which is specifically stabilized by potassium ions in vitro, has a core of three stacked guanine tetrads that is capped by two intrastrand A-T base pairs.