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.     

Active oligomeric ATP synthases in mammalian mitochondria

Recently, by analysis of mildly solubilized mitochondrial membranes new biochemical evidences were obtained for the occurrence of ATP synthase dimers in mitochondria of different eukaryotes from yeast to mammals. In the case of yeast even higher ATP synthase oligomers could be found. Here, we analysed by BN- and CN-PAGE mammalian (bovine and rat) mitochondria from five different tissues, which were efficiently but very mildly solubilized with digitonin. In mitochondria from all investigated tissues besides ATP synthase monomers (V(1)) not only dimeric ATP synthase (V(2)) but for the first time also higher oligomers, at least trimers (V(3)) and tetramers (V(4)), were separated. Compared with BN-PAGE, by CN-PAGE analysis the yields of preserved respiratory supercomplexes as well as of oligomeric ATP synthases (V(2-4)) were significantly increased. The latter represent the majority of total ATP synthases in all cases. Importantly, all different ATP synthase species from the five tissues displayed in-gel ATP hydrolase activity, suggesting that homooligomeric ATP synthases are the constitutive, enzymatically competent organization of mammalian ATP synthases in the inner mitochondrial membrane.     

A liquid medium for the production of Kabatiella zeae conidia.

A liquid medium, designated as Kabatiella zeae medium (KZM), containing 10.0 g of carboxymethylcellulose, 5.0 g of maltose, 1.5 g of peptone, 1.0 g of monobasic potassium phosphate, 1 L of distilled water, is described. Peptone was found to be the component most influential in stimulating sporulation. The optimum temperature for conidial production was 25 degrees C. In shake culture about 10(7) conidia/mL were produced in 5 days at room temperature (about 21 degrees C) when KZM was seeded with 4-day-old colony plugs. The optimum for conidial production was pH 5 with only a slight gradual reduction to pH 9. However, fungal development was abnormal at pH values of 4 and 5. Of the 29 isolates tested, only two did not sporulate in KZM or in any other medium. Because of the high yield of conidia in a short time, the ease of preparation and use, and its low cost, KZM is especially useful where large amounts of inoculum are needed.     

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.     

Supramolecular organization of cytochrome c oxidase- and alternative oxidase-dependent respiratory chains in the filamentous fungus Podospora anserina

To elucidate the molecular basis of the link between respiration and longevity, we have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This established aging model is able to respire by either the standard or the alternative pathway. In the latter pathway, electrons are directly transferred from ubiquinol to the alternative oxidase and thus bypass complexes III and IV. We show that the cytochrome c oxidase pathway is organized according to the mammalian "respirasome" model (Sch?gger, H., and Pfeiffer, K. (2000) EMBO J. 19, 1777-1783). In contrast, the alternative pathway is composed of distinct supercomplexes of complexes I and III (i.e. I(2) and I(2)III(2)), which have not been described so far. Enzymatic analysis reveals distinct functional properties of complexes I and III belonging to either cytochrome c oxidase- or alternative oxidase-dependent pathways. By a gentle colorless-native PAGE, almost all of the ATP synthases from mitochondria respiring by either pathway were preserved in the dimeric state. Our data are of significance for the understanding of both respiratory pathways as well as lifespan control and aging.     

"Respirasome"-like supercomplexes in green leaf mitochondria of spinach

Higher plant mitochondria have many unique features compared with their animal and fungal counterparts. This is to a large extent related to the close functional interdependence of mitochondria and chloroplasts, in which the two ATP-generating processes of oxidative phosphorylation and photosynthesis, respectively, take place. We show that digitonin treatment of mitochondria contaminated with chloroplasts from spinach (Spinacia oleracea) green leaves at two different buffer conditions, performed to solubilize oxidative phosphorylation supercomplexes, selectively extracts the mitochondrial membrane protein complexes and only low amounts of stroma thylakoid membrane proteins. By analysis of digitonin extracts from partially purified mitochondria of green leaves from spinach using blue and colorless native electrophoresis, we demonstrate for the first time that in green plant tissue a substantial proportion of the respiratory complex IV is assembled with complexes I and III into "respirasome"-like supercomplexes, previously observed in mammalian, fungal, and non-green plant mitochondria only. Thus, fundamental features of the supramolecular organization of the standard respiratory complexes I, III, and IV as a respirasome are conserved in all higher eukaryotes. Because the plant respiratory chain is highly branched possessing additional alternative enzymes, the functional implications of the occurrence of respiratory supercomplexes in plant mitochondria are discussed.