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International Journal of Molecular... Jul 2020For deep space exploration, reproductive health must be maintained to preserve the species. However, the mechanisms underlying the effect of changes in gravity on male...
For deep space exploration, reproductive health must be maintained to preserve the species. However, the mechanisms underlying the effect of changes in gravity on male germ cells remain poorly understood. The aim of this study was to determine the effect of simulated micro- and hypergravity on mouse sperm motility and the mechanisms of this change. For 1, 3 and 6 h, mouse sperm samples isolated from the caudal epididymis were subjected to simulated microgravity using a random position machine and 2g hypergravity using a centrifuge. The experimental samples were compared with static and dynamic controls. The sperm motility and the percentage of motile sperm were determined using microscopy and video analysis, cell respiration was determined by polarography, the protein content was assessed by Western blotting and the mRNA levels were determined using qRT-PCR. The results indicated that hypergravity conditions led to more significant changes than simulated microgravity conditions: after 1 h, the speed of sperm movement decreased, and after 3 h, the number of motile cells began to decrease. Under the microgravity model, the speed of movement did not change, but the motile spermatozoa decreased after 6 h of exposure. These changes are likely associated with a change in the structure of the microtubule cytoskeleton, and changes in the energy supply are an adaptive reaction to changes in sperm motility.
Topics: Animals; Cell Respiration; Cells, Cultured; Hypergravity; Male; Mice; Proteins; RNA, Messenger; Sperm Motility; Spermatozoa; Weightlessness; Weightlessness Simulation
PubMed: 32709012
DOI: 10.3390/ijms21145054 -
Frontiers in Oncology 2020Lactic acidosis (3 to 40 mM, pH < 6.9) is a condition found in solid tumors because tumor cells have a high rate of glucose consumption and lactate production even in...
Lactic acidosis (3 to 40 mM, pH < 6.9) is a condition found in solid tumors because tumor cells have a high rate of glucose consumption and lactate production even in the presence of oxygen; nevertheless, the microenvironment might still provide a sufficient glucose supply. Lactic acidosis has been proposed to shift metabolism from aerobic glycolysis toward oxidative phosphorylation (OXPHOS). We tested if lung tumor cells cultured under lactic acidosis shift their metabolism from glycolysis to OXPHOS by consuming extracellular lactate, increasing growth rate. We analyzed lung adenocarcinoma (A-549, A-427) cell lines and non-transformed fibroblast cells (MRC-5), which were cultured using RPMI-1640 medium initially containing lactate (2 mM) and glucose (10 mM), at pH 7.2 or 6.2 and oxygen tension 21% O (normoxia) or 2% O (hypoxia). We obtained growth curves, as well as glucose consumption and lactate production rates (measured during exponential growth) for each cell line. HIF-1α (Hypoxia-inducible factor 1 α), CS (citrate synthase) and AMPK (AMP-activated protein kinase) transcript levels were analyzed using RT-qPCR. By flow cytometry, we determined: (a) expression of glucose transporters (GLUT)1 and 4; (b) lactate transporters (MCT)1 and 4; (c) cell cycle profile, and (d) protein levels of HIF-1α, total and phosphorylated AMPK (pAMPK). Mitochondrial functionality was evaluated by measuring O consumption in tumor cells using polarography and a Clark-type electrode. Tumor and non-transformed cells used both aerobic glycolysis and OXPHOS for obtaining energy. As of 48 h of culture, lactate levels ranged from (4.5-14 mM), thus forming a lactic environment. Lactic acidosis diminished GLUT1/GLUT4 expression and glucose consumption in A-549, but not in A-427 cells, and induced differential expression of HIF-1α, AMPK, and CS transcripts. A-427 cells increased pAMPK and HIF-1α levels and shifted their metabolism increasing OXPHOS; thus supporting cell growth. Conversely, A-549 cells increased HIF-1α protein levels, but did not activate AMPK and diminished OXPHOS. A-549 cells survived by arresting cells in G1-phase. Our findings show that lactic acidosis diminishes Warburg effect in tumor cells, but this change does not necessarily promote a shift to OXPHOS. Hence, lung adenocarcinomas show a differential metabolic response even when they are under the same microenvironmental conditions.
PubMed: 32596143
DOI: 10.3389/fonc.2020.00807 -
Physiologia Plantarum Feb 2021The Mn CaO cluster of photosystem II (PSII) advances sequentially through five oxidation states (S to S ). Under the enzyme cycle, two water molecules are oxidized, O is...
The Mn CaO cluster of photosystem II (PSII) advances sequentially through five oxidation states (S to S ). Under the enzyme cycle, two water molecules are oxidized, O is generated and four protons are released into the lumen. Umena et al. (2011) have proposed that, with other charged amino acids, the R323 residue of the D1 protein could contribute to regulate a proton egress pathway from the Mn CaO cluster and Tyr via a proton channel identified from the 3D structure. To test this suggestion, a PsbA3/R323E site-directed mutant has been constructed and the properties of its PSII have been compared to those of the PsbA3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV-visible absorption spectroscopy. Neither the oscillations with a period four nor the kinetics and S-state-dependent stoichiometry of the proton release were affected. However, several differences have been found: (1) the P decay in the hundreds of ns time domain was much slower in the mutant, (2) the S Q /DCMU and S Q /DCMU radiative charge recombination occurred at higher temperatures and (3) the S Tyr , S Tyr , S Tyr split EPR signals induced at 4.2 K by visible light from the S Tyr , S Tyr , S Tyr , respectively, and the (S Tyr )' induced by NIR illumination at 4.2 K of the S Tyr state differed. It is proposed that the R323 residue of the D1 protein interacts with Tyr likely via the H-bond network previously proposed to be a proton channel. Therefore, rather than participating in the egress of protons to the lumen, this channel could be involved in the relaxations of the H-bonds around Tyr by interacting with the bulk, thus tuning the driving force required for Tyr oxidation.
Topics: Arginine; Electron Spin Resonance Spectroscopy; Oxidation-Reduction; Photosystem II Protein Complex; Protons
PubMed: 32359083
DOI: 10.1111/ppl.13115 -
Journal of Analytical Methods in... 2020products have been used for the control of diabetes (type 2) across the world. Experimental and clinical evaluations of galegine substance produced by a medicinal plant...
products have been used for the control of diabetes (type 2) across the world. Experimental and clinical evaluations of galegine substance produced by a medicinal plant () provided the pharmacological and chemical basis for metformin discovery which was confirmed for diabetes therapy. In this paper, the molecularly imprinted polymer (MIP) was synthesized for galegine, using galegine as a template molecule, methacrylic acid (MAA) as a functional monomer, ethylene glycol dimethacrylate (EGDMA) as a cross-linker, azobisisobutyronitrile (AIBN) as a reaction initiator, and acetonitrile as a solvent. The assisted functional groups, morphology, topographic image of surface, and crystalline structure of synthesized MIP were characterized by FT-IR spectroscopy, field emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM) images, and XRD diffraction pattern techniques, respectively. Also, the performance of the mentioned electrode was quantified and qualified by the differential pulse voltammetry technique (DPV). The galegine amount was determined with the polarographic technique. In this research, the galegine extraction conditions were optimized and graphene nanoparticles were used to increase the adsorption. In addition, different parameters affecting extraction were investigated such as MIP adsorbent amount, pH of solution, effect of the surfactant, and ionic compound to achieve high recovery percent. The recovery percent, limit of detection (LOD), limit of quantification (LOQ), and relative standard deviation (RSD %) were 4.101 g·mL, 12.427 g·mL, and 1.199% ( = 3), respectively. The results show that the prepared MIP can be used as an effective and inexpensive adsorbent for preconcentration and galegine extraction from a natural sample. It is noteworthy that this developed method was used successfully to determine galegine extracted from .
PubMed: 32190402
DOI: 10.1155/2020/3646712 -
Journal of Biological Inorganic... Mar 2020Oxygen affinity is an important property of metalloproteins that helps elucidate their reactivity profile and mechanism. Heretofore, oxygen affinity values were... (Review)
Review
Oxygen affinity is an important property of metalloproteins that helps elucidate their reactivity profile and mechanism. Heretofore, oxygen affinity values were determined either using flash photolysis and polarography techniques that require expensive instrumentation, or using oxygen titration methods which are erroneous at low nanomolar and at high millimolar oxygen concentrations. Here, we describe an inexpensive, easy-to-setup, and a one-pot method for oxygen affinity measurements that uses the enzyme chlorite dismutase (Cld) as a precise in situ oxygen source. Using this method, we measure thermodynamic and kinetic oxygen affinities (K and K) of different classes of heme and non-heme metalloproteins involved in oxygen transport, sensing, and catalysis. The method enables oxygen affinity measurements over a wide concentration range from 10 nM to 5 mM which is unattainable by simply diluting oxygen-saturated buffers. In turn, we were able to precisely measure oxygen affinities of a model set of eight different metalloproteins with affinities ranging from 48 ± 3 nM to 1.18 ± 0.03 mM. Overall, the Cld method is easy and inexpensive to set up, requires significantly lower quantities of protein, enables precise oxygen affinity measurements, and is applicable for proteins exhibiting nanomolar-to-millimolar affinity values.
Topics: Kinetics; Models, Molecular; Oxidoreductases; Oxygen; Thermodynamics
PubMed: 31897725
DOI: 10.1007/s00775-019-01750-6 -
Nature Communications Dec 2019In the brain, increased neural activity is correlated with increases of cerebral blood flow and tissue oxygenation. However, how cerebral oxygen dynamics are controlled...
In the brain, increased neural activity is correlated with increases of cerebral blood flow and tissue oxygenation. However, how cerebral oxygen dynamics are controlled in the behaving animal remains unclear. We investigated to what extent cerebral oxygenation varies during locomotion. We measured oxygen levels in the cortex of awake, head-fixed mice during locomotion using polarography, spectroscopy, and two-photon phosphorescence lifetime measurements of oxygen sensors. We find that locomotion significantly and globally increases cerebral oxygenation, specifically in areas involved in locomotion, as well as in the frontal cortex and the olfactory bulb. The oxygenation increase persists when neural activity and functional hyperemia are blocked, occurred both in the tissue and in arteries feeding the brain, and is tightly correlated with respiration rate and the phase of respiration cycle. Thus, breathing rate is a key modulator of cerebral oxygenation and should be monitored during hemodynamic imaging, such as in BOLD fMRI.
Topics: Animals; Brain; Cerebrovascular Circulation; Female; Hemodynamics; Humans; Locomotion; Magnetic Resonance Imaging; Male; Mice, Inbred C57BL; Olfactory Bulb; Oxygen; Respiration; Wakefulness
PubMed: 31797933
DOI: 10.1038/s41467-019-13523-5 -
Biosensors Nov 2019The benzodiazepine class of drugs are characterised by a readily electrochemically reducible azomethine group. A number are also substituted by other electrochemically... (Review)
Review
The benzodiazepine class of drugs are characterised by a readily electrochemically reducible azomethine group. A number are also substituted by other electrochemically active nitro, N-oxide, and carbonyl groups, making them readily accessible to electrochemical determination. Techniques such as polarography, voltammetry, and potentiometry have been employed for pharmaceutical and biomedical samples, requiring little sample preparation. This review describes current developments in the design and applications of electrochemical-based approaches for the determination of the benzodiazepine class of drugs form their introduction in the early 1960s to 2019. Throughout this period, state-of-the-art electroanalytical techniques have been reported for their determination. Polarography was first employed focused on mechanistic investigations. Subsequent studies showed the adsorption of many the benzodiazepines at Hg electrodes allowed for the highly sensitive technique of adsorptive stripping voltammetry to be employed. The development and introduction of other working electrode materials such as carbon led to techniques such as voltammetry to become commonly reported, and the modification of these electrodes has now become the most commonly employed approach using molecularly imprinting and nanotechnology.
Topics: Adsorption; Benzodiazepines; Biosensing Techniques; Electrochemical Techniques; Electrodes; Mercury; Molecular Structure; Surface Properties
PubMed: 31684040
DOI: 10.3390/bios9040130 -
American Journal of Physiology.... Aug 2019High energy expenditure is reported in cystic fibrosis (CF) animal models and patients. Alterations in skeletal muscle oxidative capacity, fuel utilization, and the...
High energy expenditure is reported in cystic fibrosis (CF) animal models and patients. Alterations in skeletal muscle oxidative capacity, fuel utilization, and the creatine kinase-phosphocreatine system suggest mitochondrial dysfunction. Studies were performed on congenic C57BL/6J and F508del () mice. Indirect calorimetry was used to measure gas exchange to evaluate aerobic capacity during treadmill exercise. The bioenergetic function of skeletal muscle subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) was evaluated using an integrated approach combining measurement of the rate of oxidative phosphorylation by polarography and of electron transport chain activities by spectrophotometry. CF mice have reduced maximal aerobic capacity. In SSM of these mice, oxidative phosphorylation was impaired in the presence of complex I, II, III, and IV substrates except when glutamate was used as substrate. This impairment appeared to be caused by a defect in complex V activity, whereas the oxidative system of the electron transport chain was unchanged. In IFM, oxidative phosphorylation and electron transport chain activities were preserved, whereas complex V activity was reduced, in CF. Furthermore, creatine kinase activity was reduced in both SSM and IFM of CF skeletal muscle. The decreased complex V activity in SSM resulted in reduced oxidative phosphorylation, which could explain the reduced skeletal muscle response to exercise in CF mice. The decrease in mitochondrial creatine kinase activity also contributed to this poor exercise response.
Topics: Animals; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Energy Metabolism; Female; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CFTR; Mice, Transgenic; Mitochondria, Muscle; Muscle, Skeletal; Oxidative Phosphorylation; Oxidative Stress; Physical Conditioning, Animal; Sequence Deletion
PubMed: 31211618
DOI: 10.1152/ajpendo.00064.2019