Sources and regulation of nitric oxide synthesis in uterus smooth muscle cells

It was proved that NO synthesis in isolated mitochondria of rat uterus smooth muscle depended on the entry of exogenous Ca2+ to mitochondria (inhibited by 1-10 mM Mg2+ in the absence of ATP and by 10 μM ruthenium red) and was suppressed by calmodulin antagonists (0.1-10 μM calmidazolium and 1-100 μM trifluoperazine). It was blocked by NG-nitro-L-arginine, a known antagonist of the constitutive NO-synthase, with a half-maximal inhibition effect at about 25 μM. Moderate deholesterinization of the plasma membrane of myocytes after processing with 0.01% digitonin was followed by increased nitric oxide biosynthesis by cells. The data obtained suggested that mitochondria and plasmalemma is a possible source of NO synthesis in uterine myocytes

Consideration of the contribution of chemical (non-enzymatic) conversion of substrate in the general mechanism of enzyme reaction

When enzyme-catalyzed reactions are studied, it is necessary to take into account the contribution of the chemical (non-enzymatic) conversion of the substrate to the product, which is carried out together with the enzyme-catalyzed conversion of the substrate. It is generally believed that the difference of the product concentration that was formed in the presence of the enzyme and in its absence (during the same time interval) is the concentration of the product that was formed directly in the enzyme-catalyzed reaction, i.e. that there is additivity of the product concentrations at each time point. In this paper, we have analyzed when there is additivity and how to correctly take into account the contribution of chemical (non-catalytic) substrate conversion when the enzyme-catalyzed reactions are investigated. We have shown that the additivity of product­ concentrations and initial rates is observed only for a period when the product concentration increases linear­ly with time. The longer the reaction proceeds the more the deviation from the additivity. Under equilibrium condition, there is no additivity of equilibrium product concentrations but under conditions of detailed balance the equilibrium product concentration of the overall reaction, including the enzyme-catalyzed and chemical (non-enzymatic) conversion of the substrate, is also at the same time the equilibrium concentration of the product of the enzyme-catalyzed conversion of the substrate

Obscuring the routes: confused data cannot reveal phylogeography of pea crop wild relatives (refutation to ‘Genomic diversity and macroecology of the crop wild relatives of domesticated pea’ by Smýkal et al. 2017)

Profound data confusion is reported for the paper by Smýkal et al. (Genomic diversity and macroecology of the crop wild relatives of domesticated pea. Scientific Reports 7, 17384; 2017) which challenges the validity of its scientific conclusion and the data reported

Identification of the gene coding for seed cotyledon albumin SCA in the pea (Pisum L.) genome

Albumins SCA and SAA are short, highly hydrophilic proteins accumulated in large quantities in the cotyledons and seed axes, respectively, of a dry pea (Pisum sativum L.) seed. SCA was earlier shown to have two allelic variants differing in mobility in polyacrylamide gel electrophoresis in acid medium. Using them, the corresponding gene SCA was mapped on Linkage Group V. This protein was used as a useful genetic and phylogeographical marker, which still required electrophoretic analysis of the protein while the DNA sequence of the corresponding SCA gene remained unknown. Based on the length, the positive charge under acidic conditions and the number of lysine residues of SCA and SAA albumins, estimated earlier electrophoretically, the data available in public databases were searched for candidates for the SCA gene among coding sequences residing in the region of the pea genome which, taking into account the synteny of the pea and Medicago truncatula genomes, corresponds to the map position of SCA. Then we sequenced them in a number of pea accessions. Concordance of the earlier electrophoretic data and sequence variation indicated the sequence Psat0s797g0160 of the reference pea genome to be the SCA gene. The sequence Psat0s797g0240 could encode a minor related albumin SA-a2, while a candidate gene for albumin SAA is still missing (as well as electrophoretic variation of both latter albumins). DNA amplification using original primers SCA1_3f and SCA1_3r from genomic DNA and restriction by endonuclease HindII made it possible to distinguish the SCA alleles coding for protein products with different charges without sequencing the gene. Thus, the gene encoding the highly hydrophilic albumin SCA accumulated in pea seeds, the alleles of which are useful for classification of pea wild relatives, has now been identified in the pea genome and a convenient CAPS marker has been developed on its basis

Electrochemical potential of the inner mitochondrial membrane and Ca(2+) homeostasis of myometrium cells

We demonstrated using Ca2+-sensitive fluorescent probe, mitochondria binding dyes, and confocal laser scanning microscopy, that elimination of electrochemical potential of uterus myocytes’ inner mitochondrial membrane by a protonophore carbonyl cyanide m-chlorophenyl hуdrazone (10 μM), and by a respiratory chain complex IV inhibitor sodium azide (1 mM) is associated with substantial increase of Ca2+ concentration in myoplasm in the case of the protonophore effect only, but not in the case of the azide effect. In particular, with the use of nonyl acridine orange, a mitochondria-specific dye, and 9-aminoacridine, an agent that binds to membrane compartments in the presence of proton gradient, we showed that both the protonophore and the respiratory chain inhibitor cause the proton gradient on mitochondrial inner membrane to dissipate when introduced into incubation medium. We also proved with the help of 3,3′-dihexyloxacarbocyanine, a potential-sensitive carbocyanine-derived fluorescent probe, that the application of these substances results in dissipation of the membrane’s electrical potential. The elimination of mitochondrial electrochemical potential by carbonyl cyanide m-chlorophenyl hуdrazone causes substantial increase in fluorescence of Ca2+-sensitive Fluo-4 AM dye in myoplasm of smooth muscle cells. The results obtained were qualitatively confirmed with flow cytometry of mitochondria isolated through differential centrifugation and loaded with Fluo-4 AM. Particularly, Ca2+ matrix influx induced by addition of the exogenous cation is totally inhibited by carbonyl cyanide m-chlorophenyl hydrazone. Therefore, using two independent fluorometric methods, namely confocal laser scanning microscopy and flow cytometry, with Ca2+-sensitive Fluo-4 AM fluorescent probe, we proved on the models of freshly isolated myocytes and uterus smooth muscle mitochondria isolated by differential centrifugation sedimentation that the electrochemical gradient of inner membrane is an important component of mechanisms that regulate Ca2+ homeostasis in myometrium cells

Inhibition of Na(+),K(+)-ATPase and activation of myosin ATPase by calix[4]arene C-107 cause stimulation of isolated smooth muscle contractile activity

The discovery of compounds that might modify myometrial contractility is an important area of researches. In our previous experiments, we found that some representatives of macrocyclic compounds fami­ly – calix[4]arenes – can modify the enzymatic and transport activity of membrane-bound cation-transport ATP hydrolases. The aim of this work was to study and compare the effect of calix[4]arene C-107 on the enzymatic activities of Mg2+-dependent ATPases of the uterine smooth muscle, namely: ouabain-sensitive Na+,K+-ATPase, plasma membrane Ca2+-independent “basal” Mg2+-ATPase, ATPase of the actomyosin complex and myosin subfragment-1, with effect on the contractile activity of the myometrium. It was shown that calix[4]arene C-107 efficiently inhibited myometrium Na+,K+-ATPase (I50 = 54 ± 6 nM) selectively to other ATP-hydrolases of the plasma membrane and simultaneously activated the enzymatic activity of the myosin ATPase of smooth muscles (A50 = 9.6 ± 0.7 μM). Such reciprocal biochemical effects led to the stimulation of the smooth muscle contractile activity that was demonstrated by the tensometric method using different isolated smooth muscles. Calix[4]arene С-107 was shown to stimulate the increase of the tonic component of myometrium contractions induced by oxytocin, as well as contractions of the caecum muscles induced by high-potassium solution or acetylcholine, and to maintain increased tension for a long time. Thus, calix[4]arene C-107 is a prospective compound for enhancing the smooth muscle basal tone and/or contraction in case of hypotonic dysfunctions

Тhiacalix[4]arene phosphonate C-800 as a novel fluorescent probe for zinc in living cells

Zn ions are significant for maintaining the proper human organism functioning, thus monitoring­ the zinc content in living cells and the development of sensitive tracking systems and sensors for Zn is particularly important. The purpose of the work was to study the properties of synthetic thiacalix[4]arene C-800 (5,11,17,23-tetrakis[(hydroxy-ethoxyphosphonyl)methyl])-25,26,27,28-tetrahydroxythiacalix[4]arene) as a fluo­rescent sensor for zinc ions in living cells. Our studies demonstrated that thiacalix[4]arene C-800 containing­ four hydroxy-ethoxyphosphonylmethyl groups on the upper rim exhibited fluorescent properties at 340 nm excitation wavelength. Fluorescence intensity of thiacalix[4]arene C-800 was increased significantly in the presence of Zn cations, while cations of other metals, such as Mg2+, Ca2+, Cd2+, and Pb2+ did not affect it. Computer modeling demonstrated that two Zn cations interact with the oxygen atoms of four hydroxy-ethoxyphosphonylmethyl groups. It was shown that thiacalix[4]arene C-800 quickly penetrated rat myometrial cells that led to an increased intracellular fluorescence level. The addition of Zn2+ to cells, stained with thiacalix[4]arene C-800, was followed an even greater increase of intracellular fluorescent signal intensity. No effect of thiacalix[4]arene C-800 on reactive oxygen species production in myometrial cells was detected as well as on cells viability in the range of its 50-250 μM concentrations. Thus, thiacalix[4]arene C-800 can potentially be used as a selective fluorescent probe for the detection of Zn2+ in living cells

Сalix[4]arene chalcone amides effects on myometrium mitochondria

Mitochondria are a key player in a wide range of the most important functions of the cell. Calixarenes are supramolecular compounds that have been widely used in bioorganic chemistry and biochemistry. The aim of this work was to study the effects of calix[4]arenes with two (С-1012, С-1021), three (С-1023, С-1024) and four (С-1011) chalcone amide groups on the myometrial mitochondria membranes polarization, Ca2+ concentration in the matrix of these organelles ([Ca2+]m ) and on the average hydrodynamic diameter of mitochondria. It was shown that permeabilized myometrium cells incubation with calix[4]arenes containing two or more chalcone amide groups, was accompanied by an increased level of myometrial mitochondria membranes polarization. All studied calix[4]arenes increased [Ca2+]m values in the absence and in the presence of exogenous Ca2+. The values of [Ca2+]m in the absence of exogenous Ca2+ were higher at mitochondria incubation in Mg2+-containing, than in Mg2+,ATP-containing medium. Incubation of isolated mitochondria with the studied calix[4]arenes resulted in changes of mitochondria volume: at incubation with С-1012, С-1021, C-1023 the average hydrodynamic diameter was decreased, while with С-1011 it was increased. Thus, we have shown that a short-term (5 min) incubation of mitochondria in the presence of 10 µM calix[4]arenes, which contain from two to four chalcone amide groups, increased the level of mitochondria membranes polarization, ionized Ca concentration in the matrix and had different effects on the mitochondrial volume

The plastid and mitochondrial genomes of <i>Vavilovia Formosa</i> (Stev.) Fed. and the phylogeny of related legume genera

The plastid and mitochondrial genomes of Vavilovia formosa (Stev.) Fed. were assembled on the base of the data of high-throughput sequencing of DNA isolated from a sample from North Osetia, Russia, using Illumina and PacBio platforms. The long PacBio reads were sufficient for reliable assembling organellar genomes while the short Illumina reads obtained from total DNA were unacceptable for this purpose because of substantial contamination by nuclear sequences. The organellar genomes were circular DNA molecules; the genome of mitochondria was represented by two circular chromosomes. A phylogenetic analysis on the basis of plastid genomes available in public databases was performed for some representatives of the tribes Fabeae, Trifolieae and Cicereae. As was expected, the V. formosa branch proved to be sister to the Pisum branch, and the tribe Fabeae was monophyletic. The position of Trifolium L. appeared sensitive to the phylogeny reconstruction method, either clustering with Fabeae or with the genera Medicago L., Trigonella L. and Melilotus Mill., but the internodes between successive divergences were short in all cases, suggesting that the radiation of Trifolium, other Trifolieae and Fabeae was fast, occurring within a small time interval as compared to further evolution of these lineages. The data on the relatedness of the plastid genomes of Trifolium and Fabeae correlate with the similarity of N2-fixing symbionts in these legumes represented by Rhizobium leguminosarum biovars trifolii and viciae, while the symbionts of Medicago, Melilotus and Trigonella belong to the Sinorhizobium meliloti and S. medicae species, which are distant from Rhizobium

Influence of calixarene C-90 on contractile activity of rat myometrium smooth muscles

It is known that calix[4]arene with cipher C-90 selectively and with high affinity inhibits Ca2+,Mg2+-ATPase of smooth muscle cells plasma membrane preparations. The work wis devoted to investigation of the influence of calixarene C-90 (10 µM) on spontaneous and induced (high-potassium solution and oxytocin) contractions of rat uterus longitudinal smooth muscles. Contractile activity was studied tensometrically in the isometric mode, analysis of the kinetic properties of contractions was performed by the calculation of the normalized maximal velocity of contraction (Vnс) and relaxation (Vnr) phases. Calixarene C-90 changed the spontaneous contractile activity, causing a decrease in amplitude and has no significant effect on the frequency, while slowing down of the relaxation phase of individual contractions (decreasing parameter Vnr) occurred. In the presence of non-selective NO-synthase inhibitor L-NAME (100 µM), calixarene C-90 did not cause a reduction of the amplitude of spontaneous contractions and the speed of relaxation phase returned to the control level. Furthermore, calixarene C-90 was equally contributing factor to reduced force of both oxytocin-induced (0.1 IU) and K+-induced (80 mM) contractions without affecting the nature of the increase in contractile force responses (normalized maximal velocity of contraction phase stayed at control level). The relaxation velocity of caused contractions recieved opposite changes depending on the nature of the contractile stimulation: in case of oxytocin-evoked contractions – decreased, while for K+-induced contractions – increased. In the presence of L-NAME calixarene C-90 did not cause inhibition of the maximal force K+- and oxytocin-induced contractions, but evoked changes in the kinetical para­meters of contractile responses (decrease Vnr). Thus, blocking of NO synthesis resulted in the removal of inhibiting both spontaneous and evoked contractions of smooth muscle myometrium under the influence of calixarene C-90. These results suggest that inhibition force of uterus smooth muscle contractions under the influence of calixarene C-90 is by NO-dependent way, whereas slow relaxation (decrease in normalized maximal velocity Vnr) is caused by the inhibition of Ca2+-transport function of the plasma membrane calcium pump