A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Induction of Solute Release from Nicotiana tabacum Tissue 10

For determination of the effects of polymyxin B, polymyxin E,or ethylenediamine tetra-acetic acid (EDTA) on plant cell membranes,the rates at which three solutes, K⁺, P₁, and sugar, leakedfrom treated tissue culture cell suspensions of Nicotiana tabacumwere measured. The kinetics of leakage from cells treated witheither of the polymyxins was biphasic, whereas kinetics forcells treated with EDTA was monophasic. Only K⁺ leaked frompolymyxin-treated cells during the first phase, and all threesolutes leaked during the second phase. The slower first phaseis interpreted as leakage of K⁺ from the Donnan free space andcytoplasm, and the faster second phase as the leakage of solutesfrom the vacuole. The monophasic kinetics of EDTA treatmentindicated that solutes were leaking simultaneously from cytoplasmand vacuole. Of the divalent cations tested, only Ca⁺⁺ and Mn⁺⁺counteracted the effects of polymyxin and EDTA. Ca⁺⁺ even restoredP¹ and sugar uptake. Addition of Mg⁺⁺ or Sr⁺⁺ to polymyxin-treatedcells did not stop solute leakage but actually enhanced theleakage rates. A model is presented that suggests that polymyxinor EDTA induces solute leakage by forming pores in plant cellmembranes. The effects of divalent cations on membranes oncethe pores are formed are also discussed. Key words: Polymyxin, EDTA, Nicotiana tabacum, Solute leakage     

Interaction Between Mineral Nutrients, Cytokinin and Gibberellic Acid During Growth of Sorghum at High NaCI Salinity

Sorghum bicolor (L.) Moench, cv. 610, adapted to high salinitywas able to grow at 300 mol m^–3 NaCl only when half-strengthHoagland's solution was enriched with mineral nutrients. Theoptimal growth rate was observed in full strength Hoagland'ssolution; at higher or lower concentrations growth rates werelower. In contrast, growth rate of plants exposed to 150 molm^–3 NaCl was not affected by similar modification of theHoagland solution concentration. At high salinity, additionof cytokinin (CK) or gibberellic acid (GA), or a mixture ofboth, can induce the same effect on growth as the increasedmineral nutrient concentration. Phytohormones and increasedmineral concentration have similar effects, possibly becausean imbalance in phytohormones, rather than a mineral deficiency,limits growth at 300 mol m^–3 NaCl in the presence of half-strengthHoagland solution. The change in mineral concentration in thenutrient medium, in addition to its nutritional effect, alsoapparently acts as a signal involved in hormonal balance whichallows growth at high salinity. Exposure of Sorghum to 300 molm^–3 NaCl causes a decrease in the range of nutrient concentrationswhich can sustain growth. Adjustment of the nutrient concentrationmay induce the synthesis of endogenous CK and GA concentrationsrequired for growth. In contrast, addition of CK or GA at similarconcentrations during the adaptation (pretreatment) period inhibitsgrowth and prevents the adaptation process. The response tothe exogenous phytohormone treatments depends on the time elapsedfrom the beginning of salinization. Key words: Adaptation to salinity, cytokinin, gibberellic acid, mineral nutrition, growth, Sorghum, NaCl     

Exogenous ABA as a Modulator of the Response of Sorghum to High Salinity

Treatment of Sorghum bicolor (L.) Moench, cv. 610, with abscisicacid (ABA) during the first week of sahnization with 150 molm^–3 NaCl induced enhancement of growth and acceleratedadaptation to high salinity (300 mol m^–3 NaCl) Adaptationis defined as the development of the ability of the plant tosurvive, grow, and set seeds upon exposure to a NaCl concentrationwhich is lethal for the unadapted plant In the absence of ABAthe saline pretreatment requires 20 d for the development ofadaptation (Amzallag et al., 1990), with ABA treatment the sameresult is achieved within approximately one week The exposureof the plants to non-lethal salinity (150 mol m^–3 NaCl)apparently triggers a transient sensitivity to ABA lasting forabout 8 to 10 d following the beginning of sahnization Thisperiod coincides with an increase in leaf PEP carboxylase activitywhich seems to occur faster if the plants are treated with ABA.Exogenous ABA-induced enhancement of growth and control of shootNa⁺ concentration, occur at a lower ABA concentration (10 mmolm^–3) than the induction of adaptation to salinity whichoc curs at 40 mmol m^–3 or above. The lowered shoot Na⁺concentration which is induced by a low ABA concentration isnot sufficient to induce survival of the plants in high salinity(300 mol m^–3 NaCl). Key words: Growth, adaptation to salinity, ABA     

Genome-wide differentially methylated genes in prostate cancer tissues from African-American and Caucasian men

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the β-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between β-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2′-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.