Gαo/i-stimulated proteosomal degradation of RGS20: A mechanism for temporal integration of Gs and Gi pathways

The G_s and G_i pathways interact to control the levels of intracellular cAMP. Although coincident signaling through G_s and G_i-coupled receptors can attenuate G_s-stimulated cAMP levels, it is not known if prior activation of the G_i pathway can affect signaling by G_s-coupled receptors. We have found that activated Gα_o/i interact with RGS20, a GTPase activating protein for members of the Gα_ο/i family. Interaction between Gα_o/i and RGS20 results in decreased cellular levels of RGS20. This decrease was induced by activated Gα_o and Gα_i2 but not by Gα_q, Gα_i1 or Gα_i3. The Gα_o/i-induced decrease in RGS20 can be blocked by proteasomal inhibitors lactacystin or MG132. Activated Gα_o stimulates the ubiquitination of RGS20. The serotonin-1A receptor that couples to G_o/i reduces the levels of RGS20 and this effect is blocked by lactacystin, suggesting that G_o/i promotes the degradation of RGS20. Expression of RGS20 attenuates the inhibition of β-adrenergic receptor-induced cAMP levels mediated by the serotonin-1A receptor. Prior activation of the serotonin-1A receptor results in loss of the RGS20-mediated attenuation, and the loss of attenuation is blocked when lactacystin is included during the prior treatment. These observations suggest that G_o/i-coupled receptors, by stimulating the degradation of RGS20, can regulate how subsequent activation of the G_s and G_i pathways controls cellular cAMP levels, thus allowing for signal integration.     

间隙连接及其调控因素

概述了间隙连接的结构和功能,以及pH值、电压、生长因子对它的调节作用.间隙连接作为相邻细胞间信息物质通道,起着传递细胞信息,协调细胞群体功能的作用,但是其形成过程以及对机体生理功能影响的研究有待进一步深入.pH值降低可引起间隙连接通道的关闭,电压升高降低通道的导电性,生长因子可通过影响间隙连接蛋白的形成和降解、促使间隙连接蛋白磷酸化调节其通透性.     

Effect of changes of pHi on intracellular calcium in a smooth muscle-like cell line

The effect of changes of pH_i on Ca_i were studied using fluorescent dyes in cells of the cultured smooth muscle-like line, BC3H-1. Resting Ca_i in these cells was 182 ± 12 nM (n = 74) at pH_o of 7.4. Upon exposure to NH₄Cl, which rapidly alkalinized cells, a transient increase of Cai to 349 ± 55 nM (n = 29) was observed. The peak of the transient occurred within 30 s of exposure to NH₄Cl and returned to baseline within 1 minute. Two other procedures which resulted in rapid cellular alkalinization also caused a transient rise in Ca_i: exposure to and then removal of CO₂ (Ca_i increased from 182 ± 22 to 248 ± 28 nM; n = 8); and exposure to and then removal of Na propionate (Ca_i increased from 242 ± 32 to 456 ± 71 nM; n = 9). The NH4Cl-induced Ca_i transient was eliminated by exposure to 0.2 mM TMB8 and to Ca-free solutions, but not by exposure to 0.5 mM LaCl₃. Sustained changes of pH_i can be induced by varying pH_o. When pH_o was lowered to 6.9, Ca_i fell by 49 ± 11 nM but increased by 203 ± 51 nM (n = 6) when pH_o was raised to 7.9. These data indicate that rapid alkalinization of BC3H-1 cells results in a rapid transient rise of Ca_i. This transient is most likely due to the release of Ca from intracellular stores but may also involve an increase of Ca influx. Steady state values of Cai are positively correlated with steady state pH_i. These data may have implications for the contractile state of smooth muscle during periods of acid/base disturbances and relate to the role of elevated pH_i in cells from hypertensive animals.     

Effects of potassium on the anion and cation contents of primary cultures of mouse astrocytes and neurons

Inastrocytes, as [K⁺]_o was increased from 1.2 to 10 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. As [K⁺]_o was increased from 10 to 60 mM, intracellular concentration of these three ions showed no significant change. When [K⁺]_o was increased from 60 to 122 mM, an increase in [K⁺]_i and [Cl^–]_i and a decrease in [Na⁺]_i were observed.Inneurons, as [K⁺]_o was increased from 1.2 to 2.8 mM, [Na⁺]_i and [Cl^–]_i were decreased, whereas [K⁺]_i was increased. As [K⁺]_o was increased from 2.8 to 30 mM, [K⁺]_i, [Na⁺]_i and [Cl^–]_i showed no significant change. When [K⁺]_o was increased from 30 to 122 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. Inastrocytes, pH_i increased when [K⁺]_o was increased. Inneurons, there was a biphasic change in pH_i. In lower [K⁺]_o (1.2–2.8 mM) pH_i decreased as [K⁺]_o increased, whereas in higher [K⁺]_o (2.8–122 mM) pH_i was directly related to [K⁺]_o. In bothastrocytes andneurons, changes in [K⁺]_o did not affect the extracellular water content, whereas the intracellular water content increased as the [K⁺]_o increased. Transmembrane potential (E_m) as measured with Tl-204 was inversely related to [K⁺]_o between 1.2 and 90 mM, a ten-fold increase in [K⁺]_o depolarized the astrocytes by about 56 mV and the neurons about 52 mV. The E_m values measured with Tl-204 were close to the potassium equilibrium potential (E_k) except those in neurons at lower [K⁺]_o. However, they were not equal to the chloride equilibrium potential (E_Cl) at [K⁺]_o lower than 30 mM in both astrocytes and neurons. Results of this study demonstrate that alteration of [K⁺]_o produced different changes in [K⁺]_i, [Na⁺]_i, [Cl^–]_i, and pH_i in astrocytes and neurons. The data show that astrocytes can adapt to alterations in [K⁺]_o, in such a way to maintain a more suitable environment for neurons.     

Activation of striated muscle: nearest-neighbor regulatory-unit and cross-bridge influence on myofilament kinetics

We have formulated a three-compartment model of muscle activation that includes both strong cross-bridge (XB) and Ca(2+)-activated regulatory-unit (RU) mediated nearest-neighbor cooperative influences. The model is based on the tight coupling premise--that XB retain activating Ca(2+) on the thin filament. Using global non-linear least-squares, the model produced excellent fits to experimental steady-state force-pCa and ATPase-pCa data from skinned rat soleus fibers. In terms of the model, nearest-neighbor influences over the range of Ca(2+) required for activation cause the Ca(2+) dissociation rate from regulatory-units (k(off)) to decrease and the cross-bridge association rate (f) to increase each more than ten-fold. Moreover, the rate variations occur in separate Ca(2+) regimes. The energy of activation governing f is strongly influenced by both neighboring RU and XB. In contrast, the energy of activation governing k(off) is less affected by neighboring XB than by neighboring RU. Nearest-neighbor cooperative influences provide both an overall sensitization to Ca(2+) and the well-known steep response of force to free Ca(2+). The apparent sensitivity for Ca(2+)-activation of force and ATPase is a function of cross-bridge kinetic rates. The model and derived parameter set produce simulated behavior in qualitative agreement with steady-state experiments reported in the literature for partial TnC replacement, increased [P(i)], increased [ADP], and MalNEt-S1 addition. The model is an initial attempt to construct a general theory of striated muscle activation-one that can be consistently used to interpret data from various types of muscle manipulation experiments.     

Inhibition of acetylpolyamine and spermine oxidases by the polyamine analogue chlorhexidine

Acetylpolyamine and spermine oxidases are involved in the catabolism of polyamines. The discovery of selective inhibitors of these enzymes represents an important tool for the development of novel anti-neoplastic drugs. Here, a comparative study on acetylpolyamine and spermine oxidases inhibition by the polyamine analogue chlorhexidine is reported. Chlorhexidine is an antiseptic diamide, commonly used as a bactericidal and bacteriostatic agent. Docking simulations indicate that chlorhexidine binding to these enzymes is compatible with the stereochemical properties of both acetylpolyamine oxidase and spermine oxidase active sites. In fact, chlorhexidine is predicted to establish several polar and hydrophobic interactions with the active site residues of both enzymes, with binding energy values ranging from ?7.6 to ?10.6 kcal/mol. In agreement with this hypothesis, inhibition studies indicate that chlorhexidine behaves as a strong competitive inhibitor of both enzymes, values of K_i being 0.10 μM and 0.55 μM for acetylpolyamine oxidase and spermine oxidase, respectively.     

Coeficient of relationship (RI) within and between four black venezuelan populations

The coefficient of relationship by isonymy R_i was obteined using paternal and maternal surnames of individuals in four black Venezuelan populations. The analysis of eight R_i values obtained from different combinations of surnames within each population and between population in two periods of time (1900–1920 and 1944–1967) show that: 1) Higher values of R_i are those that include combinations of maternal surnames, 2) R_i values of the second period are higher than those shown in the first, 3) No correlation was observed between R_i values and geographic distance. These results indicate that there is a tendency towards matrilocal behavior and show agreement with the degree of isolation of each population. The use of four surnames for estimating the coefficient of relationship by isonymy R_i, permits better comparisons of the relationship between and within groups and gives detailed information of the population structure.     

Characterization of the tonoplast proton pumps in Cucumis sativus L. root cells

Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifugation of microsome pellet (10,000 – 80,000 g) on discontinuous sucrose density gradient (20, 28, 32 and 42 %). Lack of PEP carboxylase (cytosol marker) and cytochrome c oxidase (mitochondrial marker) together with a slight activity of VO₄-ATPase (plasma membrane marker) and NADH-cytochrome c reductase (ER marker) in tonoplast preparation confirmed its high purity. Using latency of nitrate-inhibited ATPase and H⁺ pumping as criteria it was established that the majority of tonoplast vesicles were sealed and oriented right(cytoplasmic)-side-out. Strong acidification of the interior of vesicles observed at the presence of both, ATP and PP_i, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H⁺PP_iase. To examine and characterise of proton-transport systems in tonoplast, the effect of various inhibitors on H⁺ pumping and hydrolytic activities of ATPase and PP_iase were measured. ATP-dependent activities (H⁺ flux and ATP hydrolysis) were specifically decreased by nitrate and bafilomycin A₁, whereas the PP_iase activities were reduced in the presence of fluoride and Na⁺ ions. Both enzymes showed a similar sensitivity to DCCD and DES. The results of experiments with KCl and NaCl suggested that the vacuolar ATPase was stimulated by Cl⁻, whereas the vacuolar Pp_iase requires K⁺ ions for its activity.     

A unique mechanism of curcumin inhibition on F1 ATPase

ATP synthase (F-ATPase) function depends upon catalytic and rotation cycles of the F₁ sector. Previously, we found that F₁ ATPase activity is inhibited by the dietary polyphenols, curcumin, quercetin, and piceatannol, but that the inhibitory kinetics of curcumin differs from that of the other two polyphenols (Sekiya et al., 2012, 2014). In the present study, we analyzed Escherichia coli F₁ ATPase rotational catalysis to identify differences in the inhibitory mechanism of curcumin versus quercetin and piceatannol. These compounds did not affect the 120° rotation step for ATP binding and ADP release, though they significantly increased the catalytic dwell duration for ATP hydrolysis. Analysis of wild-type F₁ and a mutant lacking part of the piceatannol binding site (γΔ277–286) indicates that curcumin binds to F₁ differently from piceatannol and quercetin. The unique inhibitory mechanism of curcumin is also suggested from its effect on F₁ mutants with defective β–γ subunit interactions (γMet23 to Lys) or β conformational changes (βSer174 to Phe). These results confirm that smooth interaction between each β subunit and entire γ subunit in F₁ is pertinent for rotational catalysis.     

Growth and photosynthetic response of nine tropical species with long-term exposure to elevated carbon dioxide

Summary Seedlings of nine tropical species varying in growth and carbon metabolism were exposed to twice the current atmospheric level of CO₂ for a 3 month period on Barro Colorado Island, Panama. A doubling of the CO₂ concentration resulted in increases in photosynthesis and greater water use efficiency (WUE) for all species possessing C₃ metabolism, when compared to the ambient condition. No desensitization of photosynthesis to increased CO₂ was observed during the 3 month period. Significant increases in total plant dry weight were also noted for 4 out of the 5 C₃ species tested and in one CAM species, Aechmea magdalenae at high CO₂. In contrast, no significant increases in either photosynthesis or total plant dry weight were noted for the C₄ grass, Paspallum conjugatum. Increases in the apparent quantum efficiency (AQE) for all C₃ species suggest that elevated CO₂ may increase photosynthetic rate relative to ambient CO₂ over a wide range of light conditions. The response of CO₂ assimilation to internal C_i suggested a reduction in either the RuBP and/or Pi regeneration limitation with long term exposure to elevated CO₂. This experiment suggests that: (1) a global rise in CO₂ may have significant effects on photosynthesis and productivity in a wide variety of tropical species, and (2) increases in productivity and photosynthesis may be related to physiological adaptation(s) to increased CO₂.     

A new alkalitolerant <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> yeast strain is a promising model for dissecting properties and regulation of Na<Superscript>+</Superscript>-dependent phosphate transport systems

A newly isolated osmo-, salt-, and alkalitolerant Yarrowia lipolytica yeast strain is distinguished from other yeast species by its capacity to grow vigorously at alkaline pH values (9.7), which makes it a promising model organism for studying Na⁺-dependent phosphate transport systems in yeasts. Phosphate uptake by Y. lipolytica cells grown at pH 9.7 was mediated by several kinetically discrete Na⁺-dependent systems specifically activated by Na⁺. One of these, a low-affinity transporter, operated at high concentrations of extracellular phosphate. The other two, high-affinity systems, maximally active in phosphate-starved cells, were repressed or derepressed depending on the prevailing extracellular phosphate concentration and pH value. The contribution of Na⁺/P_i-cotransport systems to the total cellular phosphate uptake progressively increased with increasing pH, reaching its maximum at pH 9.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1607–1615.Original Russian Text Copyright © 2004 by Zvyagilskaya, Persson.     

Mechanism of Mss116 ATPase reveals functional diversity of DEAD-Box proteins

Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein that is essential for efficient in vivo splicing of all group I and group II introns and for activation of mRNA translation. Catalysis of intron splicing by Mss116 is coupled to its ATPase activity. Knowledge of the kinetic pathway(s) and biochemical intermediates populated during RNA-stimulated Mss116 ATPase is fundamental for defining how Mss116 ATP utilization is linked to in vivo function. We therefore measured the rate and equilibrium constants underlying Mss116 ATP utilization and nucleotide-linked RNA binding. RNA accelerates the Mss116 steady-state ATPase ∼ 7-fold by promoting rate-limiting ATP hydrolysis such that inorganic phosphate (P_i) release becomes (partially) rate-limiting. RNA binding displays strong thermodynamic coupling to the chemical states of the Mss116-bound nucleotide such that Mss116 with bound ADP-P_i binds RNA more strongly than Mss116 with bound ADP or in the absence of nucleotide. The predominant biochemical intermediate populated during in vivo steady-state cycling is the strong RNA-binding Mss116-ADP-P_i state. Strong RNA binding allows Mss116 to fulfill its biological role in the stabilization of group II intron folding intermediates. ATPase cycling allows for transient population of the weak RNA-binding ADP state of Mss116 and linked dissociation from RNA, which is required for the final stages of intron folding. In cases where Mss116 functions as a helicase, the data collectively favor a model in which ATP hydrolysis promotes a weak-to-strong RNA binding transition that disrupts stable RNA duplexes. The subsequent strong-to-weak RNA binding transition associated with P_i release dissociates Mss116-RNA complexes, regenerating free Mss116.     

Acetylcholinesterase/Butyrylcholinesterase inhibition activity of some new carbacylamidophosphate deriviatives

Eight newly synthesized carbacylamidophosphates with the general formula RC(O)NHP(O)Cl₂ with R = pCl–C₆H₄ 1a, pBr–C₆H₄ 2a, C₆H₅ 3a, and pMe–C₆H₄ 4a and RC(O)NHP(O)(NC₄H₈O)₂ R = pCl–C₆H₄ 1b, pBr–C₆H₄ 2b, C₆H₅ 3b, pMe–C₆H₄ 4b, were selected to compare the inhibition kinetic parameters, IC₅₀, K_i, k_p and K_D, on human erythrocyte acetylcholinesterase (hAChE) and bovine serum butyrylcholinesterase (BuChE), Also, the in vivo inhibition potency of compound 2a, 2b and 3a, were studied. The data demonstrates that compound 2a and compound 2b are the potent sensitive as AChE and BuChE inhibitors respectively, and the inhibition of hAChE is about 10-fold greater than that of BuChE.     

无机磷影响叠氮钠对ATP合成酶合成活性的抑制

利用ADP和放射性磷直接合成ATP的方法,研究了无机磷(P_i)和叠氮钠对猪心线粒体ATP合成酶（F₁F_O-ATPase）ATP合成活性的影响.结果发现无机磷除作为合成ATP的底物参与F₁F_O-ATPase的合成反应外,还对F₁F_O-ATPase的合成活性呈现抑制作用,在1 mmol/L ADP存在时,随着P_i浓度由0.01～10 mmol/L增加,抑制合成作用越来越强.与叠氮钠在低浓度时(小于1 mmol/L)只抑制ATP水解,不影响ATP合成的观点不同.实验结果显示0.1 mmol/L叠氮钠表观激活F₁F_O-ATPase的ATP合成活性,且激活程度与反应体系中所加P_i的浓度呈负相关.当固定P_i浓度（0.1 mmol/L）后,随着叠氮钠浓度的增加表观激活程度也在变化,叠氮钠与磷浓度相等时表观激活程度最大,直至叠氮钠浓度接近0.5 mmol/L时,开始呈现表观抑制现象,叠氮钠浓度高于1 mmol/L之后,就出现解偶联现象.     

Investigations of boron uptake at the cellular level

The efficiency of recovery of P by iron oxide-impregnated filter paper, as used in the new P_i test for soil phosphorus, was found to depend on the method used for impregnating the paper with iron oxide and could range from as little as 28% to more than 98%. The greatest efficiency of recovery was obtained with filter papers which had been washed with deionised water following iron oxide-impregnation. These filter papers were also found to give the most reproducible results. ei]{gnB E}{fnClothier}     

The growth and phosphorus utilisation of plants in sterile media when supplied with inositol hexaphosphate, glucose 1-phosphate or inorganic phosphate

Seedlings of six temperate pasture species, three grasses and three legumes, were grown for 19–24 days in sterile agar or sand-vermiculite media, in the presence of inorganic phosphate (P_i), glucose 1-phosphate (G1P) or inositol hexaphosphate (IHP). Agar (pH 5.0) had a low IHP-sorbing capacity while IHP was almost completely sorbed in sand-vermiculite. P_i and G1P were relatively available in both media. Growth of each species was measured in relation to phosphorus (P) supply and levels of P_i supply at which shoot yields reached 90% of maximum yield (P_crit) were determined. P_crit values were generally higher for the legume species than for the grasses, and were six-fold higher for Trifolium subterraneum L. seedlings when grown in sand-vermiculite relative to agar. When supplied with G1P, seedlings of the six species grew as well as plants supplied with P_i. By contrast, IHP was a poor source of P for plant growth, even when supplied in agar at levels up to 40-fold greater than P_crit. Using the growth of T. subterraneum in the presence of IHP, it was calculated that roots released approximately 0.09 nkat phytase g^-1 root dry wt per day, over 20 days of growth. By supplementing agar containing IHP with phytase from Aspergillus niger (E.C. 3.1.3.8; 0.012 nkat plant^-1, or _∼1.3 nkat g^-1 root dry wt), sufficient P became available to enable T. subterraneum seedlings to grow as well as P_i-supplied plants. These results indicate that while pasture plants can quite effectively use P from some organic P sources (e.g. G1P), the acquisition of phytate-P is limited both by availability of substrate and the capacity of plant roots to hydrolyse available IHP. This revised version was published online in June 2006 with corrections to the Cover Date.     

A study of quantitative dynamics of F-actin during oocyte maturation in the starfish Asterias amurensis

We studied the actin cytoskeleton state in Asterias amurensis oocytes within 30 min after the 1-methyladenine-induced maturation until the germinal vesicle breakdown. The total amount of actin remained unchanged during oocyte maturation. In immature oocytes, the major part of actin is not a part of filaments, but in the presence of 1-methyladenine massive actin polymerization began already within 20 min. Electron immunocytochemistry methods demonstrated joint localization of actin and α_i-protein in the cytoplasm. They were redistributed from the cortex to the cytoplasm in the presence of 1-methyladenine. A possible involvement of actin cytoskeleton in transmembrane transduction of the hormonal signal at the postreceptor stages is discussed.     

Elevated intracellular pH appears in aged oocytes and causes oocyte aneuploidy associated with the loss of cohesion in mice

Increases in the aneuploidy rate caused by the deterioration of cohesion with increasing maternal age have been well documented. However, the molecular mechanism for the loss of cohesion in aged oocytes remains unknown. In this study, we found that intracellular pH (pH_i) was elevated in aged oocytes, which might disturb the structure of the cohesin ring to induce aneuploidy. We observed for the first time that full-grown germinal vesicle (GV) oocytes displayed an increase in pH_i with advancing age in CD1 mice. Furthermore, during the in vitro oocyte maturation process, the pH_i was maintained at a high level, up to ～7.6, in 12-month-old mice. Normal pH_i is necessary to maintain protein localization and function. Thus, we put forward a hypothesis that the elevated oocyte pH_i might be related to the loss of cohesion and the increased aneuploidy in aged mice. Through the in vitro alkalinization treatment of young oocytes, we observed that the increased pH_i caused an increase in the aneuploidy rate and the sister inter-kinetochore (iKT) distance associated with the strength of cohesion and caused a decline in the cohesin subunit SMC3 protein level. Young oocytes with elevated pH_i exhibited substantially the increase in chromosome misalignment.     

Gas Exchange Responses to CO2 Concentration Instantaneously Elevated in Flag Leaves of Winter Wheat Cultivars Released in Different Years

Three winter wheat (Triticum aestivum L.) cultivars, representatives of those widely cultivated in Beijing over the past six decades, were grown in the same environmental conditions. Net photosynthetic rate (P _N) per unit leaf area and instantaneous water use efficiency (WUE) of flag leaves increased with elevated CO₂ concentration. With an increase in CO₂ concentration from 360 to 720 µmol mol^–1, P _N and WUE of Jingdong 8 (released in 1990s and having the highest yield) increased by 173 and 81 %, while those of Nongda 139 (released in 1970s) increased by 88 and 66 %, and Yanda 1817 (released in 1945, with lowest yield) by 76 and 65 %. Jingdong 8 had the highest P _N and WUE values under high CO₂ concentration, but Yanda 1817 showed the lowest P _N. Stomatal conductance (g _s) of Nongda 139 and Yanda 1817 declined with increasing CO₂ concentration, but g _s of Jingdong 8 firstly went down and then up as the CO₂ concentration further increased. Intercellular CO₂ concentration (C _i) of Jingdong 8 and Nongda 139 increased when CO₂ concentration elevated, while that of Yanda 139 increased at the first stage and then declined. Jingdong 8 had the lowest C _i of the three wheat cultivars, and Yanda 1817 had the highest C _i value under lower CO₂ concentrations. However, Jingdong 8 had the highest P _N and lowest C _i at the highest CO₂ concentration which indicates that its photosynthetic potential may be high.     

Muscle proteins — their actions and interactions

Muscle contracts by the myosin cross-bridges ‘rowing’ the actin filaments past the myosin filaments. In the past year many structural details of this mechanism have become clear. Structural studies indicate distinct states for myosin S1 in the rigor, ATP or ‘down’ conformation and in the products complex (ADP·P_i) or ‘up’ state. Crystallographic studies substantiate this classification and yield details of the transformation. The isomerization ‘up’ to ‘down’ is the power stroke of muscle. This consists in the main of large changes of angle of the ‘lever arm’ (at the distal part of the myosin head) which can account for an 11 nm power stroke.