Zur physikalischen Chemie der histologischen Färbung

Ohne ZusammenfassungDie I.–VII. Mitteilung erscheint in Folia anatomica japonica.     

Multi-dimensional Transcriptional Remodeling by Physiological Insulin In Vivo

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Characterization of the modes of binding between human sweet taste receptor and low-molecular-weight sweet compounds

One of the most distinctive features of human sweet taste perception is its broad tuning to chemically diverse compounds ranging from low-molecular-weight sweeteners to sweet-tasting proteins. Many reports suggest that the human sweet taste receptor (hT1R2-hT1R3), a heteromeric complex composed of T1R2 and T1R3 subunits belonging to the class C G protein-coupled receptor family, has multiple binding sites for these sweeteners. However, it remains unclear how the same receptor recognizes such diverse structures. Here we aim to characterize the modes of binding between hT1R2-hT1R3 and low-molecular-weight sweet compounds by functional analysis of a series of site-directed mutants and by molecular modeling-based docking simulation at the binding pocket formed on the large extracellular amino-terminal domain (ATD) of hT1R2. We successfully determined the amino acid residues responsible for binding to sweeteners in the cleft of hT1R2 ATD. Our results suggest that individual ligands have sets of specific residues for binding in correspondence with the chemical structures and other residues responsible for interacting with multiple ligands.     

Molecular modeling of SWR-0342SA, a beta3-selective agonist, with beta1- and beta3-adrenoceptor

The molecular dynamics (MD) simulations study in the formation of the complex between compound SWR-0342SA and beta-ARs suggested that upon binding SWR-0342SA stimulates receptor activation through residues network (Asp104, Leu335 in beta(1)-AR; Asp117, Ser209, Leu303, Ser191 in beta(3)-AR) in an active conformation state. The models suggest that the structural origin of the selectivity of SWR-0342SA to beta(3)-AR vs. beta(1)-AR comes from the following results: (a) the tight interaction between the agonist and the TMs 3, 5, 6 and 2 nd EC loop. Asp117 interacts with the cationic amino group of the agonist molecule. (b) Additional contacts are done with Ser209, Leu303 and Ser191. These results are in good agreement with the binding affinities (pKi values) of SWR-0342SA to beta-AR family expressed in recombinant mammalian cells.     

Ectopic expression of pumpkin gibberellin oxidases alters gibberellin biosynthesis and development of transgenic Arabidopsis plants

Immature pumpkin (Cucurbita maxima) seeds contain gibberellin (GA) oxidases with unique catalytic properties resulting in GAs of unknown function for plant growth and development. Overexpression of pumpkin GA 7-oxidase (CmGA7ox) in Arabidopsis (Arabidopsis thaliana) resulted in seedlings with elongated roots, taller plants that flower earlier with only a little increase in bioactive GA4 levels compared to control plants. In the same way, overexpression of the pumpkin GA 3-oxidase1 (CmGA3ox1) resulted in a GA overdose phenotype with increased levels of endogenous GA4. This indicates that, in Arabidopsis, 7-oxidation and 3-oxidation are rate-limiting steps in GA plant hormone biosynthesis that control plant development. With an opposite effect, overexpression of pumpkin seed-specific GA 20-oxidase1 (CmGA20ox1) in Arabidopsis resulted in dwarfed plants that flower late with reduced levels of GA4 and increased levels of physiological inactive GA17 and GA25 and unexpected GA34 levels. Severe dwarfed plants were obtained by overexpression of the pumpkin GA 2-oxidase1 (CmGA2ox1) in Arabidopsis. This dramatic change in phenotype was accompanied by a considerable decrease in the levels of bioactive GA4 and an increase in the corresponding inactivation product GA34 in comparison to control plants. In this study, we demonstrate the potential of four pumpkin GA oxidase-encoding genes to modulate the GA plant hormone pool and alter plant stature and development.     

A mimetic of the mSin3-binding helix of NRSF/REST ameliorates abnormal pain behavior in chronic pain models

The neuron-restrictive silencing factor NRSF/REST binds to neuron-restrictive silencing elements in neuronal genes and recruits corepressors such as mSin3 to inhibit epigenetically neuronal gene expression. Because dysregulation of NRSF/REST is related to neuropathic pain, here, we have designed compounds to target neuropathic pain based on the mSin3-binding helix structure of NRSF/REST and examined their ability to bind to mSin3 by NMR. One compound, mS-11, binds strongly to mSin3 with a binding mode similar to that of NRSF/REST. In a mouse model of neuropathic pain, mS-11 was found to ameliorate abnormal pain behavior and to reverse lost peripheral morphine analgesia. Furthermore, even in the less well epigenetically defined case of fibromyalgia, mS-11 ameliorated symptoms in a mouse model, suggesting that fibromyalgia is related to the dysfunction of NRSF/REST. Taken together, these findings show that the chemically optimized mimetic mS-11 can inhibit mSin3-NRSF/REST binding and successfully reverse lost peripheral and central morphine analgesia in mouse models of pain.     

Production of Dwarf Lettuce by Overexpressing a Pumpkin Gibberellin 20-Oxidase Gene

We investigated the effect of overexpressing a pumpkin gibberellin (GA) 20-oxidase gene encoding an enzyme that forms predominantly biologically inactive products on GA biosynthesis and plant morphology in transgenic lettuce (Lactuca sativa cv Vanguard) plants. Lettuce was transformed with the pumpkin GA 20-oxidase gene downstream of a strong constitutive promoter cassette (El2-35S-Omega). The transgenic plants in which the pumpkin gene was detected by polymerase chain reaction were dwarfed in the T(2) generation, whereas transformants with a normal growth phenotype did not contain the transgene. The result of Southern-blot analysis showed that the transgene was integrated as a single copy; the plants segregated three dwarfs to one normal in the T(2) generation, indicating that the transgene was stable and dominant. The endogenous levels of GA(1) and GA(4) were reduced in the dwarfs, whereas large amounts of GA(17) and GA(25), which are inactive products of the pumpkin GA 20-oxidase, accumulated in these lines. These results indicate that a functional pumpkin GA 20-oxidase is expressed in the transgenic lettuce, resulting in a diversion of the normal pathway of GA biosynthesis to inactive products. Furthermore, this technique may be useful for controlling plant stature in other agricultural and horticultural species.