Calcium-permeable AMPA and kainate receptors of GABAergic neurons.

This Commentary presents a brief discussion of the action of glutamate calcium permeable receptors present with neurons on the release of the neurotransmitter gamma-aminobutyric acid (GABA). In particular, Glutamate sensitive Kainic Acid Receptors (KARs) and α-Amino-3-hydroxy-5-Methyl-4-isoxazole Propionic Acid Receptor (AMPARs) are Na+ channels that typically cause neuronal cells to depolarize and release GABA. Some of these receptors are also permeable to Ca2+ and are hence involved in the calcium-dependent release of GABA neurotransmitters. Calcium-permeable kainate and AMPA receptors (CP-KARs and CP-AMPARs) are predominantly located in GABAergic neurons in the mature brain and their primary role is to regulate GABA release. AMPARs which do not contain the GluA2 subunit are mainly localized in the postsynaptic membrane. CP-KAR receptors are located mainly in the presynapse. GABAergic neurons expressing CP-KARs and CP-AMPARs respond to excitation earlier and faster, suppressing hyperexcitation of other neurons by the advanced GABA release due to an early rapid [Ca2+]i increase. CP-AMPARs have demonstrated a more pronounced impact on plasticity compared to NMDARs because of their capacity to elevate intracellular Ca2+ levels independently of voltage. GABAergic neurons that express CP-AMPARs contribute to the disinhibition of glutamatergic neurons by suppressing GABAergic neurons that express CP-KARs. Hence, the presence of glutamate CP-KARs and CP-AMPARs is crucial in governing hyperexcitation and synaptic plasticity in GABAergic neurons.

ANALYSIS THE RESULTS OF SURGICAL TREATMENT AND EARLY REHABILITATION OF PATIENTS WITH MASSIVE TEARS THE ROTATOR CUFF THE SHOULDER.

The aim of the study was to evaluate the treatment outcomes in patients who underwent arthroscopic repair of large and massive rotator cuff tears and underwent rehabilitation at different stages of the recovery process. The clinical study group consisted of 88 patients, with an age range of 47 to 68 years (mean age 52,7±9,5 years). All patients underwent a double-row repair of the rotator cuff tendons and tenodesis or tenotomy of the long head of the biceps tendon under arthroscopic guidance. The results were assessed using the Constant Shoulder Score and the Oxford Shoulder Score scales at 6 and 12 months after the surgery. A total of 88 patients were selected, with 50 patients in the main group (MG) who had a 6-week immobilization period. The control group (CG) consisted of 38 patients who had a shortened immobilization period of 3-4 weeks due to patient preference or recommendations from other rehabilitation centers where patients underwent rehabilitation. Shoulder immobilization was performed using a standard sling with a triangular pillow and a 15° abduction angle in the shoulder joint. Following the surgeon's recommendation, all patients were offered a rehabilitation program consisting of three periods: immobilization (0-6 weeks), functional (6-12 weeks), and training (>12 weeks) periods. Comparative analysis of the treatment results using the Oxford Shoulder Score scale showed that significantly better results were obtained in the MG patients (41,5±2,1 points) compared to the CG patients (34,2±3,6 points) at 6,2±1,2 months (p˂0.05). Comparative analysis of the treatment results using the Oxford Shoulder Score scale at 12,2±1,3 months showed that there were no statistically significant differences between the MG and CG results (MG - 44,5±2,2 and CG - 42,4±3, p>0,05). Similarly, according to the Constant Shoulder Score scale, better results were observed in the MG both at 6 months and 12 months after the surgery (excellent in 82% of MG vs 36.8% in CG, good in 18% of MG vs 57.9% in CG, respectively). Thus, in the short term, early activation of the operated joint leads to delayed healing of the operated tissues and worsening of joint function, as confirmed by the comparative analysis of the examined groups. Significant advantages of prolonged immobilization (at least 6 weeks) were found in short-term observation (up to 6 months), and no differences in functional outcomes were observed in the long term during the follow-up at 12 months.

To the Mechanism of the Antiarrhythmic Action of Compound ALM-802: the Role of Ryanodine Receptors.

The effect of the compound N1-(2,3,4-trimethoxy)-N2-{2-[(2,3,4-trimethoxybenzyl)amino]ethyl}-1,2-ethane-diamine (code ALM-802) on the amplitude of the Ca2+ response in the cell was studied in in vitro experiments. The concentration of intracellular calcium was assessed using a Fura-2 two-wave probe. The experiments were performed on a culture of isolated rat hippocampal neurons. The effect of compound ALM-802 on the activity of ryanodine receptors (RyR2) was studied on an isolated strip of rat myocardium. The compound ALM-802 (69.8 µM) in hippocampal neurons causes a significant decrease in the amplitude of the Ca2+ response induced by addition of KCl to the medium. Experiments performed on an isolated myocardial strip showed that compound ALM-802 (10-5 M) almost completely blocked the positive inotropic reaction of the strip to the RyR2 agonist caffeine (5×10-5 M). The data obtained indicate that the decrease in the concentration of Ca2+ ions in the cell caused by ALM-802 is due to its ability to block RyR2 located on the membrane of the sarcoplasmic reticulum, which can be associated with the antiarrhythmic activity of the compound.

PhthisisBioMed Artificial Medical Intelligence: Software for Automated Analysis of Digital Chest X-ray/Fluorograms.

The scope of diagnostic medical examinations increases from year to year causing a reasonable desire to develop and implement new technologies to diagnostics and medical data analysis. Artificial intelligence (AI) algorithms became one of the most promising solutions to this problem and proved themselves in the course of mass practical application. During the three-year Moscow experiment started in 2020, the possibility was achieved to develop methodologies of AI use and to successfully implement it into the regional level healthcare system. In this article, the authors share their experience in developing a medical AI service using the example of PhthisisBioMed AI service and the results of its application in real clinical activities environment. This AI service has shown its quality and reliability confirmed by technological monitoring. Clinical trials of PhthisisBioMed AI service were conducted on a specially prepared verified data set (n=1536) considering epidemiological indicators of the thoracic organs major diseases prevalence. The mean sensitivity of the service was 0.975 (95% CI: 0.966-0.984). PhthisisBioMed medical AI service is registered as a medical device (medical device registration certificate No.RZN 2022/17406 dated May 31, 2022) and is actively used in the Russian Federation as a diagnostic tool to reduce the burden on radiologists and to accelerate the process of medical report obtaining.

Analysis of Electrophysiological Mechanisms of N-Deacetyllapaconitine Monochlorhydrate, the Main Metabolite of Lappaconitine Hydrobromide.

In in vitro experiments on isolated rat hippocampal neurons, we studied the electrophysiological mechanisms of the antiarrhythmic effects of N-deacetyllappaconitine monochlorhydrate (DALCh), active metabolite of lappaconitine hydrobromide (allapinin). Electrical activity of neurons was recorded by the patch-clamp method in the whole cell configuration. It was shown that DALCh increased the duration of both slow and fast depolarization phases and decreased the amplitude of the action potential. DALCh effectively inhibited transmembrane currents of Na+ ions and partially K+ ions through the corresponding transmembrane voltage-gated ion channels. Thus, DALCh, in contrast to lappaconitine hydrobromide, belongs not to 1C, but to the 1A class of antiarrhythmics according to the Vaughan-Williams classification.

Methodology for Conducting Post-Marketing Surveillance of Software as a Medical Device Based on Artificial Intelligence Technologies.

The aim of the study was to develop a methodology for conducting post-registration clinical monitoring of software as a medical device based on artificial intelligence technologies (SaMD-AI). Materials and Methods: The methodology of post-registration clinical monitoring is based on the requirements of regulatory legal acts issued by the Board of the Eurasian Economic Commission. To comply with these requirements, the monitoring involves submission of the review of adverse events reports, the review of developers' routine reports on the safety and efficiency of SaMD-AI, and the assessment of the system for collecting and analyzing developers' post-registration data on the safety and efficiency of medical devices. The methodology was developed with regard to the recommendations of the International Medical Device Regulators Forum and the documents issued by the Food and Drug Administration (USA). Field-testing of this methodology was carried out using SaMD-AI designed for diagnostic imaging. Results: The post-registration monitoring of SaMD-AI consists of three key stages: collecting user feedback, technical monitoring and clinical validation. Technical monitoring involves routine evaluation of SaMD-AI output data quality to detect and remove flaws in a timely manner, and to secure the product stability. Major outcomes include an ordered list of technical flaws in SaMD-AI and their classification using evidence from diagnostic imaging studies. The application of this methodology resulted in a gradual reduction in the number of studies with flaws due to timely improvements in artificial intelligence algorithms: the number of flaws decreased to 5% in various aspects during subsequent testing. Clinical validation confirmed that SaMD-AI is capable of producing clinically meaningful outputs related to its intended use within the functionality determined by the developer. The testing procedure and the baseline testing framework were established during the field testing. Conclusion: The developed methodology will ensure the safety and efficiency of SaMD-AI taking into account its specifics as intangible medical devices. The methodology presented in this paper can be used by SaMD-AI developers to plan and carry out the post-registration clinical monitoring.

Mechanisms Underlying the Antiarrhythmic Action of Compound ALM-802.

The mechanisms underlying the antiarrhythmic action of compound trihydrochloride N1-(2,3,4-trimethoxy)-N2-{2-[(2,3,4-trimethoxybenzyl)amino]ethyl}-1,2-ethane-diamine (code ALM-802) were studied in vitro. The experiments were performed on a culture of rat hippocampal neurons. The electrical activity of neurons was recorded by the patch-clamp method in the whole cell configuration. It is shown that the compound ALM-802 effectively blocks potential-dependent Na+ and K+ channels and does not affect the activity of potential-dependent Ca2+ channels. The inhibition of currents through these channels is dose-dependent; the IC50 of Na+ and K+ channels were 94±4 and 67±3 µM, respectively. These findings indicate that compound ALM-802 combines the properties of class I and class III antiarrhythmic agents according to the Vaughan-Williams classification.

[CLINICAL DIAGNOSTICS OF DDH AND PECULIARITIES OF HIP JOINT DEVELOPMENT IN CHILDREN THROUGHOUT THE FIRST YEAR OF LIFE].

The aim is to determine the reliability and feasibility of using the system of selective ultrasound screening of children in groups at risk of DDH and to determine the peculiarities of the hip joints maturation. The hip joints were examined in 1539 children of the first year of life and 812 newborns. All children were divided into two groups to assess the effectiveness of the system of selective ultrasound screening of the hip joints, identified by clinical signs and risk factors DDH - with risk factors and (or) clinical signs of DDH and without risk factors and clinical signs of DDH. The study of the developmental characteristics of the hip joints is based on the analysis of sonographic indicators of the hip joints in children from birth to 10 months old. Also, to assess the hip joints at birth, all children who were examined in the maternity hospital were divided into four groups according to seasonality: those born in summer - 116, in autumn - 208, in winter - 282 and in spring - 206 people. The children underwent ultrasound examination of the hip joints according to R. Graf and determination of clinical signs and risk factors of DDH. Statistical processing was performed in Microsoft Excel and StatSoft Inc. STATISTICA. The obtained statistical data allow us to draw conclusions about the development and ossification potential of the acetabulum. A healthy hip joint has significant development potential in the first 3 months of life. The assessment of the condition of the hip joints of newborns in different seasons of the year showed that the discovered influence of seasonal factors on the development of the hip joints. Mean alpha angle values are lower in infants born in the winter and fall months and high in infants born in spring and summer. An insignificant correlation was revealed between risk factors and clinical signs of DDH and the state of development of the acetabulum in children of the first year of life. The expediency of the system of general ultrasound screening of the infants hip has been substantiated, given that when using the system of selective screening of risk groups, 12% (every eighth children) of DDH in children remains undiagnosed.

Long-term exposure to low doses of ionizing radiation and COVID-19 pandemic: oncohematological aspects.

For more than 35 years after Chornobyl catastrophe, about 5 million people in Ukraine, Republic of Belarus and Russian Federation inhabit the territories that are residually contaminated with long-lived radionuclides such as 137Cs, 90Sr. The previous studies of the Reference Laboratory operating at RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology allowed specifying the effects of the protracted low dose irradiation on the state of the hematopoietic and lymphoid tissues resulting in the increased proportion of the B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma and acute myeloid leukemia among the patients referred from the contaminated areas of Ukraine. Since the beginning of 2020, these effects of radiation were superimposed by the factors associated with COVID-19 pandemic. SARS-CoV-2 infection is associated with the significant impact on hematopoiesis and immune system. Particular attention should be given to the role of such combined burden in the development of the immunodeficiency-associated lymphoid neoplasms. The extensive studies of the combined effects of low dose irradiation and COVID-19 within the large affected populations could be made a priority in future endeavors of epidemiologists and oncohematologists.

Involvement of NMDA and GABA(A) receptors in modulation of spontaneous activity in hippocampal culture: Interrelations between burst firing and intracellular calcium signal.

Spontaneous burst firing is a hallmark attributed to the neuronal network activity. It is known to be accompanied by intracellular calcium [Са2+]i oscillations within the bursting neurons. Studying mechanisms underlying regulation of burst firing is highly relevant, since impairment in neuronal bursting accompanies different neurological disorders. In the present study, the contribution of NMDA and GABA(A) receptors to the shape formation of spontaneous burst -was studied in cultured hippocampal neurons. A combination of inhibitory analysis with simultaneous registration of neuronal bursting by whole-cell patch clamp and calcium imaging was used to assess spontaneous burst firing and [Са2+]i level. Using bicuculline and D-AP5 we showed that GABA(A) and NMDA receptors effectively modulate burst plateau phase and [Са2+]i transient spike which can further affect action potential (AP) amplitudes and firing frequency within a burst. Bicuculline significantly elevated the amplitude and reduced the duration of both burst plateau phase and [Са2+]i spike resulting in an increase of AP firing frequency and shortening of AP amplitudes within a burst. D-AP5 significantly decreases the amplitude of both plateau phase and [Са2+]i spike along with a burst duration that correlated with an increase in AP amplitudes and reduced firing frequency within a burst. The effect of bicuculline was occluded by co-addition of D-AP5 revealing modulatory role of GABA(A) receptors to the NMDA receptor-mediated formation of the burst. Our results provide new evidence on importance of NMDA and GABA(A) receptors in shaping burst firing and Ca2+transient spikes in cultured hippocampal neurons.

Potential mechanism of GABA secretion in response to the activation of GluK1-containing kainate receptors.

Hippocampal GABAergic neurons are subdivided into more than 20 subtypes that are distinguished by features and functions. We have previously described the subpopulation of GABAergic neurons, which can be identified in hippocampal cell culture by the calcium response to the application of domoic acid (DoA), an agonist of kainate receptors (KARs). Here, we investigate the features of DoA-sensitive neurons and their GABA release mechanism in response to KARs activation. We demonstrate that DoA-sensitive GABAergic neurons express GluK1-containing KARs because ATPA, a selective agonist of GluK1-containing receptors, induces the calcium response exclusively in these GABAergic neurons. Our experiments also show that NASPM, previously considered a selective antagonist of calcium-permeable AMPARs, blocks calcium-permeable KARs. We established using NASPM that GluK1-containing receptors of the studied population of GABAergic neurons are calcium-permeable, and their activation is required for GABA release, at least in particular synapses. Notably, GABA release occurs even in the presence of tetrodotoxin, indicating that propagation of the depolarizing stimulus is not required for GABA release in this case. Thus, our data demonstrate that the activation of GluK1-containing calcium-permeable KARs mediates the GABA release by the studied subpopulation of GABAergic neurons.

Chest MRI of patients with COVID-19.

During the pandemic of novel coronavirus infection (COVID-19), computed tomography (CT) showed its effectiveness in diagnosis of coronavirus infection. However, ionizing radiation during CT studies causes concern for patients who require dynamic observation, as well as for examination of children and young people. For this retrospective study, we included 15 suspected for COVID-19 patients who were hospitalized in April 2020, Russia. There were 4 adults with positive polymerase chain reaction (PCR) test for COVID-19. All patients underwent magnetic resonance imaging (MRI) examinations using MR-LUND PROTOCOL: Single-shot Fast Spin Echo (SSFSE), LAVA 3D and IDEAL 3D, Echo-planar imaging (EPI) diffusion-weighted imaging (DWI) and Fast Spin Echo (FSE) T2 weighted imaging (T2WI). On T2WI changes were identified in 9 (60,0%) patients, on DWI - in 5 (33,3%) patients. In 5 (33,3%) patients lesions of the parenchyma were visualized on T2WI and DWI simultaneously. At the same time, 4 (26.7%) patients had changes in lung tissue only on T2WI. (P(McNemar) = 0,125; OR = 0,00 (95%); kappa = 0,500). In those patients who had CT scan, the changes were comparable to MRI. The results showed that in case of CT is not available, it is advisable to conduct a chest MRI for patients with suspected or confirmed COVID-19. Considering that T2WI is a fluid-sensitive sequence, if imaging for the lung infiltration is required, we can recommend the abbreviated MRI protocol consisting of T2 and T1 WI. These data may be applicable for interpreting other studies, such as thoracic spine MRI, detecting signs of viral pneumonia of asymptomatic patients. MRI can detect features of viral pneumonia.

[Artificial intelligence for diagnosis of vertebral compression fractures using a morphometric analysis model, based on convolutional neural networks].

BACKGROUND: Pathological low-energy (LE) vertebral compression fractures (VFs) are common complications of osteoporosis and predictors of subsequent LE fractures. In 84% of cases, VFs are not reported on chest CT (CCT), which calls for the development of an artificial intelligence-based (AI) assistant that would help radiology specialists to improve the diagnosis of osteoporosis complications and prevent new LE fractures. AIMS: To develop an AI model for automated diagnosis of compression fractures of the thoracic spine based on chest CT images. MATERIALS AND METHODS: Between September 2019 and May 2020 the authors performed a retrospective sampling study of ССТ images. The 160 of results were selected and anonymized. The data was labeled by seven readers. Using the morphometric analysis, the investigators received the following metric data: ventral, medial and dorsal dimensions. This was followed by a semiquantitative assessment of VFs degree. The data was used to develop the Comprise-G AI mode based on CNN, which subsequently measured the size of the vertebral bodies and then calculates the compression degree. The model was evaluated with the ROC curve analysis and by calculating sensitivity and specificity values. RESULTS: Formed data consist of 160 patients (a training group - 100 patients; a test group - 60 patients). The total of 2,066 vertebrae was annotated. When detecting Grade 2 and 3 maximum VFs in patients the Comprise-G model demonstrated sensitivity - 90,7%, specificity - 90,7%, AUC ROC - 0.974 on the 5-FOLD cross-validation data of the training dataset; on the test data - sensitivity - 83,2%, specificity - 90,0%, AUC ROC - 0.956; in vertebrae demonstrated sensitivity - 91,5%, specificity - 95,2%, AUC ROC - 0.981 on the cross-validation data; for the test data sensitivity - 79,3%, specificity - 98,7%, AUC ROC - 0.978. CONCLUSIONS: The Comprise-G model demonstrated high diagnostic capabilities in detecting the VFs on CCT images and can be recommended for further validation.

Taxifolin protects neurons against ischemic injury in vitro via the activation of antioxidant systems and signal transduction pathways of GABAergic neurons.

Cerebral blood flow disturbances lead to the massive death of brain cells. The death of >80% of cells is observed in hippocampal cell cultures after 40 min of oxygen and glucose deprivation (ischemia-like conditions, OGD). However, there are some populations of GABAergic neurons which are characterized by increased vulnerability to oxygen-glucose deprivation conditions. Using fluorescent microscopy, immunocytochemical assay, vitality tests and PCR-analysis, we have shown that population of GABAergic neurons are characterized by a different (faster) Ca2+ dynamics in response to OGD and increased basal ROS production under OGD conditions. A plant flavonoid taxifolin inhibited an excessive ROS production and an irreversible cytosolic Ca2+ concentration increase in GABAergic neurons, preventing the death of these neurons and further excitation of a neuronal network; neuroprotective effect of taxifolin increased after incubation of 24 h and correlated with increased expression of antiapoptocic and antioxidant genes Stat3 Nrf-2 Bcl-2, Bcl-xL, Ikk2, and genes coding for AMPA and kainate receptor subunits; in addition, taxifolin decreased expression of prooxidant enzyme NOS and proinflammatory cytokine IL-1ß.

Aminoethane Sulfonic Acid Magnesium Salt Inhibits Ca2+ Entry Through NMDA Receptor Ion Channel In Vitro.

The effect of a cerebroprotective agent magnesium bis-aminoethanesulfonate (laboratory code FS-LKhT-317) on intracellular calcium concentration was studied by the fluorescent imaging technique on neuroglial cell culture from Spraque-Dawley rat hippocampus. The substance produced a pronounced inhibitory effect and suppressed NMDA receptor activity in concentrations of ≥50 µM. The observed effects were reversible or partially reversible and were detected by a decrease in Ca2+ signal amplitude in neurons in response to NMDA applications in a Mg2+-free medium and by inhibition of Ca2+ pulses in magnesium-free medium (elimination of magnesium block).

[DUAL PROAPOPTOTIC AND PRONECROTIC EFFECT OF HYDROGEN PEROXIDE ON HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS].

The ratio of early apoptosis and late apoptosis (necrosis) in the cultured human umbilical vein endothelial cells was estimated after exposure to hydrogen peroxide (H2O2) in vitro trying to keep them close to the physiological conditions (high cell density, high serum content, H2O2 concentration not over 500 µM). Cell viability was assessed using flow cytometry and simultaneous staining with fluorescent dyes PO-PRO-1 to detect early apoptotic cells, and DRAQ7 to detect late apoptotic and necrotic cells. The data obtained suggest that the primary mechanism of cytotoxic response is apoptosis. The critical concentration of H2O2 causing the death of the cell population in a dense monolayer is 250 µM. Lower concentrations of H2O2 (up to 200 µM) cause death of individual cells; however, viability of endothelial cell population is retained, and response to calcium activating agonists does not change compared with control cells.

Metagenomic Analysis of the Dynamic Changes in the Gut Microbiome of the Participants of the MARS-500 Experiment, Simulating Long Term Space Flight.

A metagenomic analysis of the dynamic changes of the composition of the intestinal microbiome of five participants of the MARS-500 experiment was performed. DNA samples were isolated from the feces of the participants taken just before the experiment, upon 14, 30, 210, 363 and 510 days of isolation in the experimental module, and two weeks upon completion of the experiment. The taxonomic composition of the microbiome was analyzed by pyrosequencing of 16S rRNA gene fragments. Both the taxonomic and functional gene content of the microbiome of one participant were analyzed by whole metagenome sequencing using the SOLiD technique. Each participant had a specific microbiome that could be assigned to one of three recognized enterotypes. Two participants had enterotype I microbiomes characterized by the prevalence of Bacteroides, while the microbiomes of two others, assigned to type II, were dominated by Prevotella. One participant had a microbiome of mixed type. It was found that (1) changes in the taxonimic composition of the microbiomes occurred in the course of the experiment, but the enterotypes remained the same; (2) significant changes in the compositions of the microbiomes occurred just 14-30 days after the beginning of the experiment, presumably indicating the influence of stress factors in the first stage of the experiment; (3) a tendency toward a reversion of the microbiomes to their initial composition was observed two weeks after the end of the experiment, but complete recovery was not achieved. The metagenomic analysis of the microbiome of one of the participants showed that in spite of variations in the taxonomic compositions of microbiomes, the "functional" genetic composition was much more stable for most of the functional gene categories. Probably in the course of the experiment the taxonomic composition of the gut microbiome was adaptively changed to reflect the individual response to the experimental conditions. A new, balanced taxonomic composition of the microbiome was formed to ensure a stable gene content of the community as a whole without negative consequences for the health of the participants.

[Regulation of potential-dependant calcium channels by 5-HT1B serotonin receptors in various populations of hippocampal cells].

Metabotropic serotonin receptors of 5HT1-type in brain neurons participate in regulation of such human emotional states as aggression, fear and dependence on alcohol. Activated presynaptic 5-HT1B receptors suppress the Ca2+ influx through the potential-dependent calcium channels in certain neurons. The Ca2+ influx into the cells has been measured by increase of calcium ions concentration in cytoplasm in reply to the depolarization caused by 35mM KC1. Using system of image analysis in hippocampal cells culture we found out that Ca2+-signals to depolarization oin various populations of neurons differed in form, speed and amplitude. 5HT1B receptor agonists in 86 +/- 3 % of neurons slightly suppressed the activity of potential-dependent calcium channels. Two minor cell populations (5-8 % of cells each) were found out, that strongly differed in Ca2+ signal desensitization. Calcium signal caused by depolarization in one cells population differed in characteristic delay and high rate of decay. 5HT1B receptor agonists strongly inhibited the amplitude of the Ca2+ response on KCl only in this population of neurons. The calcium signal in second cell population differed by absence desensitization and smaller amplitude which constantly increased during depolarization. 5HT 1 B receptor agonists increased the calcium response amplitude to depolarization in this population of neurons. Thus we show various sensitivity of potential-dependent calcium channels of separate neurons to 5HTB1 receptor agonist.

Burst of succinate dehydrogenase and α-ketoglutarate dehydrogenase activity in concert with the expression of genes coding for respiratory chain proteins underlies short-term beneficial physiological stress in mitochondria.

Conditions for the realization in rats of moderate physiological stress (PHS) (30-120 min) were selected, which preferentially increase adaptive restorative processes without adverse responses typical of harmful stress (HST). The succinate dehydrogenase (SDH) and α-ketoglutarate dehydrogenase (KDH) activity and the formation of reactive oxygen species (ROS) in mitochondria were measured in lymphocytes by the cytobiochemical method, which detects the regulation of mitochondria in the organism with high sensitivity. These mitochondrial markers undergo an initial 10-20-fold burst of activity followed by a decrease to a level exceeding the quiescent state 2-3-fold by 120 min of PHS. By 30-60 min, the rise in SDH activity was greater than in KDH activity, while the activity of KDH prevailed over that of SDH by 120 min. The attenuation of SDH hyperactivity during PHS occurs by a mechanism other than oxaloacetate inhibition developed under HST. The dynamics of SDH and KDH activity corresponds to the known physiological replacement of adrenergic regulation by cholinergic during PHS, which is confirmed here by mitochondrial markers because their activity reflects these two types of nerve regulation, respectively. The domination of cholinergic regulation provides the overrestoration of expenditures for activity. In essence, this phenomenon corresponds to the training of the organism. It was first revealed in mitochondria after a single short-time stress episode. The burst of ROS formation was congruous with changes in SDH and KDH activity, as well as in ucp2 and cox3 expression, while the activity of SDH was inversely dependent on the expression of the gene of its catalytic subunit in the spleen. As the SDH activity enhanced, the expression of the succinate receptor decreased with subsequent dramatic rise when the activity was becoming lower. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaption and therapy.

An expression system for regulated protein production in Synechocystis sp. PCC 6803 and its application for construction of a conditional knockout of the ferrochelatase enzyme.

Synechocystis sp. PCC 6803 is a model organism for the study of photosynthetic processes. Methods to genetically manipulate this bacterium are essential to investigate these processes and to evaluate potential biotechnological applications. We developed a vector for controllable expression of proteins using a platform for stable integration of the expression cassette into the genome. The respective gene is translationally fused to the promoter of the petJ gene encoding cytochrome c(553) that is repressed by copper. Maximal expression from this promoter is achieved under copper depletion, whereas normal copper concentrations in standard medium lead to low expression rates. We show here the application of this system for construction of a conditional knockout mutant for the ferrochelatase, which is an essential enzyme in heme biosynthesis. Using different amounts of copper in the medium we were able to control the amount of ferrochelatase in the cell resulting in a varying expression of the phenotype.