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In the title complex, C(6)F(6) x C(14)H(12), nearly parallel molecules of trans-stilbene and librationally disordered hexafluorobenzene form a mixed stack, with each molecule lying on an independent inversion centre. Adjacent stacks pack together in a herring-bone manner.
A male cone-specific promoter from Pinus radiata D. Don (radiata pine) was used to express a stilbene synthase gene (STS) in anthers of transgenic Nicotiana tabacum plants, resulting in complete male sterility in 70% of transformed plants. Three plants were 98%-99.9% male sterile, as evidenced by pollen germination. To identify the stage at which transgenic pollen first developed abnormally, tobacco anthers from six different developmental stages were assayed microscopically. Following the release of pollen grains from tetrads, transgenic pollen displayed an increasingly flake-like structure, which gradually rounded up during the maturation process. We further investigated whether STS expression may have resulted in an impaired flavonol or sporopollenin formation. A specific flavonol aglycone stain was used to demonstrate that significant amounts of these substances were produced only in late stages of normal pollen development, therefore excluding a diminished flavonol aglycone production as a reason for pollen ablation. A detailed analysis of the exine layer by transmission electron microscopy revealed minor structural changes in the exine layer of ablated pollen, and pyrolysis-gas chromatography-mass spectroscopy indicated that the biochemistry of sporopollenin production was unaffected. The promoter-STS construct may be useful for the ablation of pollen formation in coniferous gymnosperms and male sterility may potentially be viewed as a prerequisite for the commercial use of transgenic conifers.
Chalcone synthase (CHS) and stilbene synthase (STS) catalyze different cyclization reactions of the common tetraketide to give different products, naringenin chalcone and resveratrol, respectively. We have previously observed in vitro cross-reaction of CHS and STS overexpressed in Escherichia coli, resveratrol production by CHS and chalcone production by STS. When expressed in eucaryotic cells, or in E. coli as thioredoxin-fusion proteins, CHS and STS exhibited reduced cross-reaction. STS refolded from inclusion bodies also showed reduced cross-reaction. While addition of bovine serum albumin and pH in the reaction were without noticeable effect, addition of glycerol decreased the cross-reaction of CHS likely due to its stabilizing effect on enzyme conformation. These results were interpreted to provide supporting evidence to our earlier proposition (Yamaguchi T. et al., FEBS Lett., 460, 457-4 61 (1999)) that the in vitro cross-reaction of CHS and STS is due to intrinsic capability of these enzymes to catalyze different types of cyclization, which, in turn, is endowed by conformational flexibility of their active sites.
We examined the effects of chlorpromazine on NO(3)(-) transport between erythrocytes (RBCs) and extracellular fluid. Chlorpromazine (10 microg/ml) did not influence NO(3)(-) movement in both whole blood and RBC suspension. Though an anion exchanger (AE1) inhibitor DIDS (4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, 100 microM) did not alter NO(3)(-) movement in whole blood, it inhibited the movement in a concentration-dependent manner in the RBC suspension. The inhibition was abrogated by plasma and albumin concentration-dependently. Our results indicated that chlorpromazine had no effect on NO(3)(-) transport through AE1 and that the inertness of DIDS on AE1 in whole blood is due to interference by albumin in plasma.
It is known that the subcutaneous injection of a single dose of sodium selenite into suckling rats results in the development of large nuclear opacities. The intracellular transport of selenite in various cells, except lens cells, occurs via the Cl/HCO3 exchanger. The aim of the present study is to investigate the possible role of the anion-exchange inhibitor, disulfonic stilbene (SITS), in the selenite-induced catarogenesis in the rat pups. Wistar albino rats (8-10 d old) were separated into three groups: one control and two experimental. The first experimental group was injected subcutaneously with a single dose of 30 nmol sodium selenite/g body weight. The second experimental group was injected with a single dose of 10 nmol SITS/g body weight 15 min before the same dose selenite injection. The control group did not have any injections. The stage of cataract development was examined on d 7 postinjection with slit-lamp photographs. In SITS pretreated group, all eyes remained transparent (considered as stage 0), whereas in the selenite-injected group, the animals did have different stage of nuclear cataract; 8 animals have stage 5, 10 animals have stage 4, and 4 animals have stage 3. A pretreatment of SITS completely prevented cataract formation of the selenite-induced cataract model in rat pups.
Two new aspects of mammalian porin are presented. First, by affinity chromatography we show that channel active human or bovine porin reversibly bind the stilbene-disulfonate group of the chloride channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). The procedure is suitable for further purification of porin after enrichment by ion exchange chromatography and shows a yield of 24.3%. The data support our recent proposal that VDAC forms part of the ORDIC channel complex which is affected in cystic fibrosis. Second, a purification scheme for mammalian porin is given starting with direct solubilisation of ground bovine skeletal muscle to avoid breaking up tissue. About 130 mg of channel active "Porin 31BM" are enriched from 946 g muscle tissue. Concerning its apparent molecular mass, primary structure, channel activity, channel conductance and voltage dependence the molecule shows high similarity to human porin. "Porin 31BM" is furthermore labelled by antibodies raised against human B lymphocyte derived "Porin 31HL".
To investigate whether or not MDCK cells may be used as a model for beta-intercalated cells, we studied: (1) The effect of luminal [Cl-]0 changes on pHi measured by video-imaging micro-fluorometry, (2) the influence of the inhibitor 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) on anion-exchange activity, and (3) the effect of acetazolamide on intracellular pH-indicator (c-SNAFL-2) accumulation and anion-exchange activity. At least three different modes of fluorescence accumulation were found in confluent monolayers: cells with high, low or undetectable fluorescence. Highly fluorescent cells responded to a rise of [Cl-]0 (30-140 mM) with a proportional decrease of pHi (7.6-6.4). Acetazolamide (10(-4) M) completely blocked the acidifying effects of the increased [Cl-]0, indicating that HCO3- is the intracellular ion exchanged for extra-cellular Cl-. Acetazolamide caused a reduction of SNAFL-2 fluorescence suggesting that carbonic anhydrase activity contributes to indicator accumulation. The high DIDS concentration (50 microM) required to prevent intracellular acidification suggests that the exchanger involved is identical to that present in beta-intercalated cells. All cells of non-confluent monolayers were highly fluorescent and expressed Cl-/ HCO3(-)-exchanger activity. In conclusion, highly fluorescent MDCK cells in confluent monolayers have a luminal DIDS inhibitable, carbonic anhydrase dependent Cl-/HCO3(-)-exchanger, and may therefore be used as a model for beta-intercalated cells.
Thermal activation is not required for barrier crossing reactions in the photoisomerization of cis-stilbene, as demonstrated by computer simulations. The activation is achieved by using the excess energy from the photoexcitation. Moreover, the reaction proceeds with large energy transfers but small conformational changes. The reaction pathway is influenced by these effects and also by the solvent.
Chalcone synthase (CHS) and stilbene synthase (STS) catalyse condensation reactions of p-coumaroyl-CoA and three C(2) units from malonyl-CoA up to a common tetraketide intermediate but then catalyse different cyclization reactions to produce naringenin chalcone and resveratrol respectively. On the basis of sequence alignment with other condensing enzymes including 3-ketoacyl-(acyl carrier protein) synthases of polyketide and fatty-acid synthases, site-directed mutagenesis was performed on the active-site G(372)FGPG loops in CHS and STS. The CHS-P375G mutant showed a 6-fold decrease in overall condensing activity with selectively increased production of p-coumaroyltriacetic acid lactone (CTAL, the derailment product of the tetraketide intermediate). Meanwhile, resveratrol production by STS-P(375)G strongly decreased to give various products in the order CTAL> resveratrol approximately bisnoryangonin>naringenin. As a result, naringenin production (cross-reaction) by STS-P(375)G was close to 30% of resveratrol production. Both G(374)L mutants of CHS and STS showed no condensing activity with residual malonyl-CoA decarboxylase activity. These results suggested that the G(372)FGPG loop in CHS and STS contribute to a determination of the outcome during cyclization reactions by serving as a part of the active-site scaffold on which the stereochemistry of cyclization is performed. These observations provide the first biochemical indication that cyclization reactions are modulated by active-site geometry. The implications for the evolutionary relationship of these enzymes are also discussed.
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