Prediction of the changes in thermodynamic stability of proteins caused by single amino acid substitutions

The functional (synthetic) activity of blood lymphocytes and bone marrow hematopoietic cells in ground squirrels was studied in different seasons and at different stages of the torpor-arousal cycle. The effect of γ-irradiation on animals in different physiological states was also studied. The synthetic activity of cells was estimated from the amount of active RNA per unit DNA in the cell (parameter α). The α values in lymphocytes were minimal in hibernating animals (January–March), reached a peak upon their complete awakening (April), slightly decreased in the summer activity period, and decreased further in the prehibernation autumn period (November). During winter arousals between torpor bouts, this parameter reached the same values as in summer. The dynamics of parameter α in bone marrow hematopoietic cells were generally similar: minimal values in November and higher between torpor bouts than in summer. The peak of synthetic activity of proliferating hematopoietic cells recorded upon awakening from hibernation in April was mainly due to the accumulation of cells in the G₁ and G₂ phases of the cell cycle, and its decrease in summer reflected prevalent transition from G₂ to mitosis and then partly to G₀. In the torpor-arousal-euthermia cycle, two stages of awakening were distinguished, differing considerably in most of the test parameters. The synthetic activity and the total number of blood and bone marrow cells in ground squirrels irradiated in the state of torpor did not differ significantly from those in nonirradiated torpid animals. The adverse effect of radiation in animals irradiated at the initial stage of awakening was lesser than in animals irradiated in the active state, whereas animals at the second stage of awakening proved more vulnerable to acute irradiation. The physiological state of ground squirrels exposed to ionizing radiation at different phases of the torpor-arousal-euthermia cycle plays a key role in the dynamics of qualitative and quantitative characteristics of blood system cells. The results of this study indicate that the hypometabolic state of ground squirrels during hibernation is a factor of protection from the impact of ionizing radiation on the whole body and on the immune system in particular.     

The awakening of dormant neuronal precursors in the adult and aged brain

Beyond the canonical neurogenic niches, there are dormant neuronal precursors in several regions of the adult mammalian brain. Dormant precursors maintain persisting post-mitotic immaturity from birth to adulthood, followed by staggered awakening, in a process that is still largely unresolved. Strikingly, due to the slow rate of awakening, some precursors remain immature until old age, which led us to question whether their awakening and maturation are affected by aging. To this end, we studied the maturation of dormant precursors in transgenic mice (DCX-CreER^T2/flox-EGFP) in which immature precursors were labelled permanently in vivo at different ages. We found that dormant precursors are capable of awakening at young age, becoming adult-matured neurons (AM), as well as of awakening at old age, becoming late AM. Thus, protracted immaturity does not prevent late awakening and maturation. However, late AM diverged morphologically and functionally from AM. Moreover, AM were functionally most similar to neonatal-matured neurons (NM). Conversely, late AM were endowed with high intrinsic excitability and high input resistance, and received a smaller amount of spontaneous synaptic input, implying their relative immaturity. Thus, late AM awakening still occurs at advanced age, but the maturation process is slow.     

The influence of heterogeneity on the analysis of sleep-wake architecture in the single-prolonged stress rats

Objective: To observe the influence of heterogeneity on sleep-wake architecture in single-prolonged stress(SPS) animal model. Methods: SPS rats were subdivided into low responders(LR) and high responders(HR) based on their freezing responses to a novel environment. Sleeping time(ST), awakening numbers(AN), brief awakening numbers(b AN) and frequency distribution of sleep bouts were used as observing indicators, single factor variance analysis combined with Dunnett t test were used to compare the differences between control, exposure, LR and HR groups. Results: We found sleeping time was increased only in HR group. Moreover, awakening numbers and brief awakening number increased in exposure group and HR group during the light phase, but not in LR group. The number of sleep bouts for the ranges of 40-80 s increased obviously in HR group, but not in exposure and LR group. In addition, there were significant correlation between sleep-related parameters and freezing in HR group, but not in LR group. Conclusion: Heterogeneity existed in SPS model in view of different sleep-wake architectures of SPS rats. Rats in HR group exactly mimicked the freezing response and sleep disorders of PTSD. So HR rats were more appropriate to be used as PTSD-like models, especially when studying sleep disorder in PTSD.     

Role of the PSII-H subunit in photoprotection: novel aspects of D1 turnover in Synechocystis 6803

Photosystem I-less Synechocystis 6803 mutants carrying modified PsbH proteins, derived from different combinations of wild-type cyanobacterial and maize genes, were constructed. The mutants were analyzed in order to determine the relative importance of the intra- and extramembrane domains of the PsbH subunit in the functioning of photosystem (PS) II, by a combination of biochemical, biophysical, and physiological approaches. The results confirmed and extended previously published data showing that, besides D1, the whole PsbH protein is necessary to determine the correct structure of a QB/herbicide-binding site. The different turnover of the D1 protein and chlorophyll photobleaching displayed by mutant cells in response to photoinhibitory treatment revealed for the first time the actual role of the PsbH subunit in photoprotection. A functional PsbH protein is necessary for (i) rapid degradation of photodamaged D1 molecules, which is essential to avoid further oxidative damage to the PSII core, and (ii) insertion of newly synthesized D1 molecules into the thylakoid membrane. PsbH is thus required for both initiation and completion of the repair cycle of the PSII complex in cyanobacteria.     

Firm but slippery attachment of Deinococcus geothermalis

Bacterial biofilms impair the operation of many industrial processes. Deinococcus geothermalis is efficient primary biofilm former in paper machine water, functioning as an adhesion platform for secondary biofilm bacteria. It produces thick biofilms on various abiotic surfaces, but the mechanism of attachment is not known. High-resolution field-emission scanning electron microscopy and atomic force microscopy (AFM) showed peritrichous adhesion threads mediating the attachment of D. geothermalis E50051 to stainless steel and glass surfaces and cell-to-cell attachment, irrespective of the growth medium. Extensive slime matrix was absent from the D. geothermalis E50051 biofilms. AFM of the attached cells revealed regions on the cell surface with different topography, viscoelasticity, and adhesiveness, possibly representing different surface layers that were patchily exposed. We used oscillating probe techniques to keep the tip-biofilm interactions as small as possible. In spite of this, AFM imaging of living D. geothermalis E50051 biofilms in water resulted in repositioning but not in detachment of the surface-attached cells. The irreversibly attached cells did not detach when pushed with a glass capillary but escaped the mechanical force by sliding along the surface. Air drying eliminated the flexibility of attachment, but it resumed after reimmersion in water. Biofilms were evaluated for their strength of attachment. D. geothermalis E50051 persisted 1 h of washing with 0.2% NaOH or 0.5% sodium dodecyl sulfate, in contrast to biofilms of Burkholderia cepacia F28L1 or the well-characterized biofilm former Staphylococcus epidermidis O-47. Deinococcus radiodurans strain DSM 20539(T) also formed tenacious biofilms. This paper shows that D. geothermalis has firm but laterally slippery attachment not reported before for a nonmotile species.     

Physiologic functions of vitamin D3 and its metabolism in the body in normal states and in some pathologies

In the review there are some results of investigations of the laboratory summarized on the mechanisms of vitamin D3 metabolism and its functioning in organism; investigation of infringement of mineral, lipid and protein metabolic processes with vitamin D3 deficiency and with different pathologies and the ways of their regulation as well as the data about some new high-effective forms of vitamin D3 for their implementation in medicine and agriculture.     

Probable role of dopamine in the regulation of ventral nephrocyte functioning in Drosophila melanogaster adults

Filtration of haemolymph in insects to remove waste products is performed by nephrocytes, which comprise accessory cells of the circulatory system that are not connected to Malpighian tubules. There are two types of nephrocytes in Drosophila: ventral cells, situated around the junction between the cardia and the oesophagus, and pericardial cells, situated around the heart. In the present study, the expression of dopamine D1 (DopR) and D2‐like (DD2R) receptors in the ventral nephrocytes of Drosophila melanogaster Meigen (Diptera: Drosophilidae) is investigated. Immunohistochemical staining with polyclonal antibodies against DopR and DD2R demonstrates the presence of these receptors in adult nephrocytes. The functioning of D. melanogaster nephrocytes is investigated by evaluation of mortality rates in flies treated with silver nitrate (AgNO₃) compared with untreated controls. To determine whether a change in the level of dopamine receptors has an effect on the functioning of nephrocytes, the antisense suppressor of DD2R gene together with a nephrocyte‐specific driver is used in the UAS‐GAL4 system. The suppression of DD2R in nephrocytes results in a significant decrease of mortality under toxic conditions. Taken together, the data obtained in the present study indicate that dopamine takes part in the control of ventral nephrocyte functioning in D. melanogaster.     

Effects of nighttime low frequency noise on the cortisol response to awakening and subjective sleep quality

The effects of night-time exposure to traffic noise (TN) or low frequency noise (LFN) on the cortisol awakening response and subjective sleep quality were determined. Twelve male subjects slept for five consecutive nights in a noise-sleep laboratory. After one night of acclimatisation and one reference night, subjects were exposed to either TN (35dB L(Aeq), 50dB L(Amax)) or LFN (40dB L(Aeq)) on alternating nights (with an additional reference night in between). Salivary free cortisol concentration was determined in saliva samples taken immediately at awakening and at three 15-minute intervals after awakening. The subjects completed questionnaires on mood and sleep quality. The awakening cortisol response on the reference nights showed a normal cortisol pattern. A significant interaction between night time exposure and time was found for the cortisol response upon awakening. The awakening cortisol response following exposure to LFN was attenuated at 30 minutes after awakening. Subjects took longer to fall asleep during exposure to LFN. Exposure to TN induced greater irritation. Cortisol levels at 30 minutes after awakening were related to "activity" and "pleasantness" in the morning after exposure to LFN. Cortisol levels 30 minutes after awakening were related to sleep quality after exposure to TN. This study thus showed that night time exposure to LFN may affect the cortisol response upon wake up and that lower cortisol levels after awakening were associated with subjective reports of lower sleep quality and mood.     

Levels and interactions of p27, cyclin D3, and CDK4 during the formation and maintenance of the corpus luteum in mice

The cyclin-dependent kinase (CDK) inhibitor p27, the regulator of the cell cycle, is required for proper functioning of luteinizing/luteinized cells in vivo. Since different members of the CDK family may be targeted by p27 during luteinization-associated cell cycle exit, this in vivo study further analyzed the organization of the network of cell cycle regulators that may underlie both the establishment and maintenance of the luteal phenotype. Most importantly, it shows that the luteinization process is associated with down-regulation of CDK2 and cyclin D1, and up-regulation of p27 and cyclin D3. Both p27 and cyclin D3 proteins not only accumulated during initial phases of luteinization, but they remained elevated until termination of the luteal function. Along with its accumulation, p27 lost physical contact with CDK2 and instead became associated with CDK4. In fully luteinized cells, all cyclin D3 was incorporated into complexes with p27, some complexes being p27/cyclin D3/CDK4 trimers. Despite the significant amounts of CDK4 and CDK6, only nonphosphorylated forms of retinoblastoma protein were detectable in fully luteinized cells. Together, our data indicate that while inhibition of proliferation is underlaid by the progressive loss of positive regulators of the cell cycle, including cyclins and CDK2, maintenance of the luteal phenotype is driven by up-regulated levels of p27 and cyclin D3, at least partially owing to formation of p27/cyclin D3/CDK4 trimers.     

Exploration of the awakening cortisol response in relation to diurnal cortisol secretory activity

Adrenocortical activity can be assessed by measurement of free cortisol in saliva. Cortisol status has important health implications in both physical and psychological terms. Assessment of cortisol status is complicated by the marked diurnal cortisol cycle. This cycle is characterised by an increase in secretory activity following awakening to achieve the morning acrophase. Thereafter it falls with a declining trend over the remainder of the day. For between subject studies the timing of sampling in relation to this cycle is an important consideration. We report a comprehensive study of the diurnal free cortisol cycle designed to analyse its components and to investigate their reliability and inter-relatedness. We instructed 42 healthy volunteers to collect saliva samples at home on two consecutive days. On each day the first sample was collected immediately upon awakening, followed by a further three samples at 15-minute intervals which collectively comprised the "awakening samples". A further four sample's were collected through the day at 3-hour intervals, all synchronized to awakening time. The cortisol response to awakening was calculated in two ways. Overall cortisol production in the first 45 minutes after awakening was calculated as area under the cortisol curve with reference to zero (AUC). The dynamic of the cortisol response to awakening was calculated as area under the cortisol response curve (AURC) with reference to the first awakening sample. In addition the underlying cortisol secretory activity was assessed based upon the diurnal three-hourly samples. All three parameters of adrenocortical activity showed reasonable stability across the two sampling days indicating all were reliable indexes of trait characteristic. AUC was representative of underlying diurnal activity but AURC was not. Measurement at any time point, 3, 6, 9 or 12 hours post-awakening was representative of the underlying 12-hour diurnal activity.     

Evidence for the presence of an F-type ATP synthase involved in sulfate respiration in Desulfovibrio vulgaris

Using a library of genomic DNA from Desulfovibrio vulgaris Miyazaki F, a strict anaerobe, and two synthetic deoxyoligonucleotide probes designed for F-type ATPases, the genes for open reading frames (ORFs) 1 to 5 were cloned and sequenced. The predicted protein sequences of the gene products indicate that they are composed of 172, 488, 294, 471, and 134 amino acids, respectively, and that they share considerable identity at the amino acid level with delta, alpha, gamma, beta, and epsilon subunits found in other F-type ATPases, respectively. Furthermore, a component carrying ATPase activity was partially purified from the cytoplasmic membrane fraction of the D. vulgaris Miyazaki F cells. The N-terminal amino acid sequences of three major polypeptides separated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis were identical to those of the products predicted by the sequences of ORF-2, ORF-3, and ORF-4, suggesting that an F-type ATPase is functioning in the D. vulgaris Miyazaki F cytoplasmic membrane. The amount of the F-type ATPase produced in the D. vulgaris Miyazaki F cells is similar to that in the Escherichia coli cells cultured aerobically. It indicates that the enzyme works as an ATP synthase in the D. vulgaris Miyazaki F cells in connection with sulfate respiration.     

Seasonal changes in the endocrine cells of the gastric epithelium of Rana temporaria

By means of the light and electron microscopy methods histotopography, quantity and ultramicroscopic organization of endocrine cells (EC) have been studied in gastric epithelium of the frog (Rana temporaria) during various periods of its life: hibernation, awakening, active life. At all the periods of life 8 types of EC are revealed. Depending on the season, certain changes both in their amount and ultrastructural organization are observed. Decreasing amount of EC is noted during hibernation and their increase in spring during awakening and in summer. Quantity of enterochromaffin cells is not subjected to seasonal fluctuations. This is explained by the fact that in hibernation these cells are also at their active functional state, since they produce serotonin necessary for maintaining deep hypothermia, specific for hibernating animals.     

Seasonal dynamics of the gastric APUD cell count in a hibernator

Light microscopy was applied to study variation in the composition and number of apudocytes in the gastric epithelium of red-cheeked sousliks over time. During hibernation, the content of serotonin-producing EC cells was elevated. This is in agreement with serotonin involvement in the maintenance of the hibernation state. The number of non-EC cells taking part in digestion, which does not occur during hibernation, was lowered. Spring awakening was accompanied with an increase in the number of non-EC cells and the lack of significant changes in cells. In souslics, which were active in winter, the content of non-cells was high, since the animals continued feeding on, and the content of EC-cells was elevated similarly to that in hibernating animals, which could be explained by genetic dependence. The number of apudocytes in the gastric mucosa was discovered to correlate in the main with the features of their function at different seasons.     

Versatile role for hnRNP D isoforms in the differential regulation of cytoplasmic mRNA turnover

An important emerging theme is that heterogeneous nuclear ribonucleoproteins (hnRNPs) not only function in the nucleus but also control the fates of mRNAs in the cytoplasm. Here, we show that hnRNP D plays a versatile role in cytoplasmic mRNA turnover by functioning as a negative regulator in an isoform-specific and cell-type-dependent manner. We found that hnRNP D discriminates among the three classes of AU-rich elements (AREs), most effectively blocking rapid decay directed by class II AREs found in mRNAs encoding cytokines. Our experiments identified the overlapping AUUUA motifs, one critical characteristic of class II AREs, to be the key feature recognized in vivo by hnRNP D for its negative effect on ARE-mediated mRNA decay. The four hnRNP D isoforms, while differing in their ability to block decay of ARE-containing mRNAs, all potently inhibited mRNA decay directed by another mRNA cis element that shares no sequence similarity with AREs, the purine-rich c-fos protein-coding region determinant of instability. Further experiments indicated that different mechanisms underlie the inhibitory effect of hnRNP D on the two distinct mRNA decay pathways. Our study identifies a potential mechanism by which cytoplasmic mRNA turnover can be differentially and selectively regulated by hnRNP D isoforms in mammalian cells. Our results support the notion that hnRNP D serves as a key factor broadly involved in general mRNA decay.     

Variation in the oxytocin receptor gene influences neurocardiac reactivity to social stress and HPA function: a population based study

Oxytocin (OT) is a nonapeptide neurohormone that is involved in a broad array of physiological and behavioral processes related to health including hypothalamic-pituitary-adrenal (HPA) axis functioning, autonomic nervous system (ANS) activity and social behaviors. The present study sought to explore the influence of genetic variation in the oxytocin receptor (SNP; rs53576) on autonomic and neurohormonal functioning across both resting and psychological stress conditions in a population based sample of older adults. Results revealed that A carrier males showed higher levels of resting sympathetic cardiac control as compared to their G/G counter parts. However, G/G participants displayed significantly higher levels of sympathetic reactivity to psychological stress with G/G males showing the highest levels of sympathetic response to stress. Although no significant effects were detected for heart rate or parasympathetic cardiac control across resting and stress conditions, results revealed that G/G participants generally displayed heightened stroke volume and cardiac output reactivity to the psychological stressor. Furthermore, analysis of diurnal fluctuations in salivary cortisol revealed that G/G participants displayed lower awakening cortisol levels and less variation in salivary cortisol across the day as compared to A carrier individuals.     

Peroxisomes exist in growth cones and move anterogradely and retrogradely in neurites of PC12D cells

Localization and movement of peroxisomes have been investigated in neurites of a subline of PC12 pheochromocytoma cells (PC12D cells). The cells were transfected with a construct encoding the green fluorescent protein and bearing the C-terminal peroxisomal targeting signal 1 SKL motif (-Ser-Lys-Leu-COOH). Peroxisomes were detected as green punctate fluorescent signals. Many peroxisomes were observed in neurites of PC12D cells, especially in neural terminal-like structures, growth cones, varicosities, and branch points. Growth cones containing many peroxisomes were active, since they extended several long filopodias. Existence of peroxisomes in growth cones and neuronal terminal-like structures suggests that peroxisomes might have some role in neuronal extension and nerve terminal functioning. Peroxisomal motility was analyzed by time-lapse imaging using a fluorescence microscope at 25 degrees C. Peroxisomes were transported bidirectionally in neurites, i.e., through anterograde and retrograde transport. This result suggests that peroxisomes move to growth cones and neural terminals from the PC12D cell body, play some role in these parts, and go back to cell body.     

Three-dimentional organization of synapses and astroglia in the hippocampus of rats and ground squirrels: new structural and functional paradigms of the synapse function

The literature data and our own data on the synaptic plasticity and remodeling of synaptic organelles in the central nervous system are reviewed. Modern techniques of laser scanning confocal microscopy and serial thin sectioning for in vivo and in vitro studies of dendritic spines, including the relationship between morphological changes and the efficacy of synaptic transmission, are discussed using, in particular, a model of long-term potentiation. The organization of dendritic spines and postsynaptic densities of different categories as well as the role of filopodia in spine genesis were analyzed. It was shown that the method of serial ultrathin sections is the most effective for unbiased quantitative stereological analysis and 3D reconstructions. By using the refined method of serial ultrathin sections with subsequent three-dimensional reconstructions, the presence of giant mitochondria in hippocampal neuronal dendrites was demonstrated. It was shown that smooth endoplasmic reticulum forms a unified continuum with the outer membrane of the mitochondrial envelope within dendrites. It was suggested that this continuum provides calcium tunneling, which makes possible intracellular signal transduction during synaptic transmission. Evidence is presented indicating the presence of gap junctions ("electrical synapses") in the synapses of mammalian brain, as well as between glial processes, and between glial cells and neurons. Our data and the data of other authors show that glial cell processes form a structural and functional glial network, which modulates the functioning of the neuronal network. The connection of dendritic spines with the glial network is shown on 3D reconstructions by analyzing the neuropil volume in CA1 hippocampal area of ground squirrels in three functional states: normothermia, provoked arousal, and hibernation when brain temperature falls below 6 degrees C. The own data of the authors are discussed indicating the formation of more than five presynaptic boutons (multiple synapses) on both CA1 mushroom-like dendritic spines and CA3 thorny excrescences. On the basis of the analysis, new ideas of the organization and functioning of synapses were suggested.     

Electron microscopic characteristics of neurosecretory cells in the preoptic nucleus of sturgeon fry]

In the sturgeon fry nucleus preopticus (Npo), light and weakly differentiated neurosecretory cells (NSC) as well as differentiated (i-cells) were revealed. Among these types of cells, there were the transitional forms. No "picnomorphic" cells were found in any Npo zone. NSC contained different amounts of elementary secretory granules 110-270 nm in diameter. The distribution of organelles was evaluated statistically, their number increasing from i-cells to the NSC. These data suggest that growth and differentiation of the NSC take place during this stage of ontogeny. The results are discussed in terms of specific functioning and maturity of the NSC.     

Smooth Muscle-Like Tissue Constructs with Circumferentially Oriented Cells Formed by the Cell Fiber Technology

The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments.