Photoinhibition and zeaxanthin formation in intact leaves : a possible role of the xanthophyll cycle in the dissipation of excess light energy

Comparative studies of chlorophyll a fluorescence, measured with a pulse amplitude modulated fluorometer, and of the pigment composition of leaves, suggest a specific role of zeaxanthin, a carotenoid formed in the xanthophyll cycle, in protecting the photosynthetic apparatus against the adverse effects of excessive light. This conclusion is based on the following findings: (a) exposure of leaves of Populus balsamifera, Hedera helix, and Monstera deliciosa to excess excitation energy (high light, air; weak light, 2% O₂, 0% CO₂) led to massive formation of zeaxanthin and a decrease in violaxanthin. Over a wide range of conditions, there was a linear relationship between either variable, F_v, or maximum fluorescence, F_m, and the zeaxanthin content of leaves. (b) When exposed to photoinhibitory light levels in air, shade leaves of H. helix had a higher capacity for zeaxanthin formation, at the expense of β-carotene, than shade leaves of M. deliciosa. Changes in fluorescence characteristics suggested that, in H. helix, the predominant response to high light was an increase in the rate of nonradiative energy dissipation, whereas, in M. deliciosa, photoinhibitory damage to photosystem II reaction centers was the prevailing effect. (c) Exposure of a sun leaf of P. balsamifera to increasing photon flux densities in 2% O₂ and 0% CO₂ resulted initially in increasing levels of zeaxanthin (matched by decreases in violaxanthin) and was accompanied by fluorescence changes indicative of increased nonradiative energy dissipation. Above the light level at which no further increase in zeaxanthin content was observed, fluorescence characteristics indicated photoinhibitory damage. (d) A linear relationship was obtained between the ratio of variable to maximum fluorescence, F_v/F_m, determined with the modulated fluorescence technique at room temperature, and the photon yield of O₂ evolution, similar to previous findings (O Björkman, B Demmig 1987 Planta 170: 489-504) on chlorophyll fluorescence characteristics at 77 K and the photon yield of photosynthesis.     

Towards elucidating the energy of the first excited singlet state of xanthophyll cycle pigments by X-ray absorption spectroscopy

The first excited singlet state (S₁) of carotenoids (also termed 2A_g⁻) plays a key role in photosynthetic excitation energy transfer due to its close proximity to the S₁ (Q_y) level of chlorophylls. The determination of carotenoid 2A_g⁻ energies by optical techniques is difficult; transitions from the ground state (S₀, 1A_g⁻) to the 2A_g⁻ state are forbidden (“optically dark”) due to parity (g ← //→ g) as well as pseudo-parity selection rules (− ← //→ −). Of particular interest are S₁ energies of the so-called xanthophyll-cycle pigments (violaxanthin, antheraxanthin and zeaxanthin) due to their involvement in photoprotection in plants. Previous determinations of S₁ energies of violaxanthin and zeaxanthin by different spectroscopic techniques vary considerably. Here we present an alternative approach towards elucidation of the optically dark states of xanthophylls by near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The indication of at least one π* energy level (about 0.5 eV below the lowest 1B_u⁺ vibronic sublevel) has been found for zeaxanthin. Present limitations and future improvements of NEXAFS to study optically dark states of carotenoids are discussed. NEXAFS combined with simultaneous optical pumping will further aid the investigation of these otherwise hardly accessible states.     

Hydrogen peroxide induces vessel occlusions and stimulates sesquiterpenes accumulation in stems of Aquilaria sinensis

Agarwood is highly valuable resinous and fragrant heartwood, produced principally from tropical tree species in the genus Aquilaria, which is used widely in countries of the Middle East, Southeast Asia and Japan. Generally, healthy trees will not produce agarwood, but wounding of the tree initiates the production of agarwood. In this study, the pruning of actively growing saplings of Aquilaria sinensis resulted in hydrogen peroxide (H₂O₂) burst, which was followed by formation of vessel occlusions and sesquiterpene biosynthesis in the pruned stems. Treatment of the pruned stems with scavenger of H₂O₂ (ascorbate, AsA) greatly reduced the amount of H₂O₂ released, the number of vessel occlusions, and the amount of sesquiterpenes produced. In addition, exogenous H₂O₂ also induced A. sinensis plants to form vessel occlusions and produce sesquiterpenes as pruning treatment. The results indicated that H₂O₂ may be an important post-wounding signal in A. sinensis that leads to the induction of vessel occlusions formation and sesquiterpene biosynthesis, and thus H₂O₂ might play a vital role in agarwood formation in pruned stems of A. sinensis.     

High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. I. The hydrogen bonded protons of the "charge relay" system

High resolution proton nuclear magnetic resonance has been used to observe protons at the active site of chymotrypsin A_δ and at the same region of chymotrypsinogen A. A single resonance with the intensity of one proton is located in the low field region of the nuclear magnetic resonance spectrum. This resonance is observed in H₂O solutions but not in ²H₂O. On going from low to high pH the resonance titrates upfield 3 parts per million in both proteins and has a pK of 7.5. The titration can be prevented by alkylating His57 with either of two active site directed chloromethyl ketones. Using these data the proton resonance has been assigned to a proton in a hydrogen bond between His57 and Asp102. Further confirmation of this assignment lies in the observation of a similar resonance in this same low field region of the nuclear magnetic resonance spectrum of trypsin, trypsinogen, subtilisin BPN′ and α-lytic protease all of which have the Asp-His-Ser triad at their active sites.This proton resonance in chymotrypsin A_δ was used as a probe to monitor the charge state of the active site upon formation of a stable acyl-enzyme analogue N²(N-acetylalanyl)-N¹benzoylcarbazoyl-chymotrypsin A_δ. In this derivative the His-Asp proton resonance titrates from the same low pH end point as in the native enzyme, ?18 parts per million, to a new high pH end point of ?14.4 parts per million (versus ?15.0 parts per million in the native enzyme). The difference of 0.6 parts per million in the high pH end points between the native and acyl enzyme is interpreted as supporting the suggestion that a hydrogen bond exists between Ser195 and His57 in the native enzyme and zymogen.We conclude from these studies that the charge relay system from Asp102 across His57 to Ser195 is intact in chymotrypsin A_δ and chymotrypsinogen A, and that, in the native enzyme, it slightly polarizes Ser195.     

Metabolism and binding properties of 24,24-difluoro-25-hydroxyvitamin D3

To evaluate possible functional roles for 24,25-dihydroxyvitamin D₃, 24,24-difluoro-25-hydroxyvitamin D₃ has been synthesized and shown to be equally as active as 25-hydroxyvitamin D₃ in all known functions of vitamin D. The use of the difluoro compound for this purpose is based on the assumption that the C-F bonds are stable in vivo and that the fluorine atom does not act as hydroxyl in biological systems. No 24,25-dihydroxyvitamin D₃ was detected in the serum obtained from vitamin D-deficient rats that had been given 24,24-difluoro-25-hydroxyvitamin D₃, while large amounts were found when 25-hydroxyvitamin D₃ was given. Incubation of the 24,24-difluoro compound with kidney homogenate prepared from vitamin D-replete chickens failed to produce 24,25-dihydroxyvitamin D₃, while the same preparations produced large amounts of 24,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. Kidney homogenate prepared from vitamin D-deficient chickens produced 24,24-difluoro-1,25-dihydroxyvitamin D₃ from 24,24-difluoro-25-hydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. In binding to the plasma transport protein for vitamin D compounds, 24,24-difluoro-25-hydroxyvitamin D₃ is less active than 25-hydroxyvitamin D₃ and 24R,25-dihydroxyvitamin D₃. In binding to the chick intestinal cytosol receptor, 24,24-difluoro-25-hydroxyvitamin D₃ is more active than 25-hydroxyvitamin D₃ which is itself more active than 24R,25-dihydroxyvitamin D₃. The 24,24-difluoro-1,25-dihydroxyvitamin D₃ is equal to 1,25-dihydroxyvitamin D₃, and both are 10 times more active than 1,24R,25-trihydroxyvitamin D₃ in this system. These results provide strong evidence that the C-24 carbon of 24,24-difluoro-25-hydroxyvitamin D₃ cannot be hydroxylated in vivo, and, further, the 24-F substitution acts similar to H and not to OH in discriminating binding systems for vitamin D compounds.     

Effect of outer sphere anions on the structure and color of nitrosylpentaamminechromium complex

Three kinds of crystalline compounds containing the nitrosylpentaamminechromium complexes [Cr(NO)(NH₃)₅]²⁺(A) were obtained: chloride ACl₂ (red-orange), chloride perchlorate ACl(ClO₄) (brown), and perchlorate A(ClO₄)₂ (green). The cause of the color change of the complex A with the change of outer sphere anions was sought using X-ray structural data of ACl₂, ACl(ClO₄), and A(ClO₄)₂. Crystal data: ACl₂, orthorhombic, space group Cmcm, a=10.0236 (9) Å, b=9.098 (3) Å, c=10.357(1) Å, V=944.5 (5) ų, Z=4; ACl(ClO₄), tetragonal, space group P4/nmm, a=7.6986 (8) Å, c=9.9566(8) Å, V=590.1 (1) Å^3,Z=2; A(ClO₄)₂, orthorhombic, space group Pnma, a=15.760 (2) Å, b=11.480(2) Å, c=7.920 (2) Å, V=1432.9 (4) ų, Z=4. The complex cation in ACl₂ has a distorted octahedral structure with a linear CrNO moiety. The short CrN (nitrosyl) distance of 1.692 (7) Å indicates the presence of multiple bonding between the chromium atom and the nitrogen atom in the nitrosyl group. The interatomic distances and angles within the complex cations hardly change with the change of the counter anions, while the distances between the complex cations in each crystal increase in the order ACl₂<ACl(ClO₄)<A(ClO₄)₂. The bulky perchlorate anions seems to separate the complex cations, while smaller chloride anions are not large enough to separate them. The distance (3.213(5) Å) between O(NO) and N(NH₃ in the adjacent complex cation) is rather short in the crystal of ACl₂, and there are six hydrogen bonds, where the NO group is surrounded by four NH₃ ligands. The distance (4.002(5) Å) between O(NO) and N(NH₃) is much longer in the crystal of A(ClO₄)₂, indicating the presence of no hydrogen bonding. In the crystal of ACl(ClO₄) the distance (3.452(4) Å) between O(NO) and N(NH₃) is in between those of ACl₂ and A(ClO₄)₂. The presence of hydrogen bonding between O(NO) and N(NH₃ in the adjacent complex cation) seems to cause the color change with the change of outer sphere anions.     

Polymorphism of Alzheimer's Aβ17-42 (p3) Oligomers: The Importance of the Turn Location and Its Conformation

Aβ_17-42 (so-called p3) amyloid is detected in vivo in the brains of individuals with Alzheimer's disease or Down's syndrome. We investigated the polymorphism of Aβ_17-42 oligomers based on experimental data from steady-state NMR measurements, electron microscopy, two-dimensional hydrogen exchange, and mutational studies, using all-atom molecular-dynamics simulation with explicit solvent. We assessed the structural stability and the populations. Our results suggest that conformational differences in the U-turn of Aβ_17-42 lead to polymorphism in β-sheet registration and retention of an ordered β-strand organization at the termini. Further, although the parallel Aβ_17-42 oligomer organization is the most stable of the conformers investigated here, different antiparallel Aβ_17-42 organizations are also stable and compete with the parallel architectures, presenting a polymorphic population. In this study we propose that 1), the U-turn conformation is the primary factor leading to polymorphism in the assembly of Aβ_17-42 oligomers, and is also coupled to oligomer growth; and 2), both parallel Aβ_17-42 oligomers and an assembly of Aβ_17-42 oligomers that includes both parallel and antiparallel organizations contribute to amyloid fibril formation. Finally, since a U-turn motif generally appears in amyloids formed by full proteins or long fragments, and since to date these have been shown to exist only in parallel architectures, our results apply to a broad range of oligomers and fibrils.     

Zeaxanthin Synthesis, Energy Dissipation, and Photoprotection of Photosystem II at Chilling Temperatures

When leaves of a mangrove, Rhizophora mangle, were exposed to an excess of light at chilling temperatures, synthesis of zeaxanthin through violaxanthin de-epoxidation as well as nonphotochemical fluorescence quenching were markedly reduced. The results suggest a protective role of energy dissipation against the adverse effects of high light and chilling temperatures: leaves of R. mangle that had been preilluminated in 2% O₂, 0% CO₂ at low photon flux density and showed a high level of zeaxanthin, and leaves that had been kept in the dark and contained no zeaxanthin, were both exposed to high light and chilling temperatures (5°C leaf temperature) in air and then held under control conditions in low light in air at 25°C. Measurements of chlorophyll a fluorescence at room temperature showed that the photochemical efficiency of PSII and the yield of maximum fluorescence of the preilluminated leaf recovered completely within 1 to 3 hours under the control conditions. In contrast, the fluorescence responses of the predarkened leaf in high light at 5°C did not recover at all. During a dark/light transient in 2% O₂, 0% CO₂ in low light at 5°C, nonphotochemical fluorescence quenching increased linearly with an increase in the zeaxanthin content in leaves of R. mangle. In soybean (Glycine max) leaves, which contained a background level of zeaxanthin in the dark, a similar treatment with excess light induced a level of nonphotochemical fluorescence quenching that was not paralleled by an increase in the zeaxanthin content.     

A comprehensive study on the 5-hydroxytryptamine3A receptor binding of agonists serotonin and m-chlorophenylbiguanidine

Serotonin type 3 receptors (5-HT₃R) are members of the ligand gated ion channel receptor family. In this study, the interactions of the agonists serotonin (5-HT) and m-chlorophenylbiguanidine (mCPBG) at the binding site of the 5-HT_3AR were investigated at an atomic level. Site-directed mutagenesis studies in Loop B and E along with our earlier published results from mutations within Loops A, C, and D provide comprehensive data on the interaction of 5-HT and mCPBG with 5-HT_3ARs. Using this data we have constructed a refined homology model of the 5-HT_3AR that considers all of the available experimental data. 5-HT and mCPBG were docked into the newly constructed homology model and the amino acid residues critical in binding of these agonists were compared and analyzed. Our docking results reveal many similar binding interactions for 5-HT and mCPBG. Namely, residues THR181, TRP183, PHE226, ILE228, TYR234 and GLU129 were all found to play key roles in binding of both 5-HT and mCPBG. However, the results also revealed two important differences that exist between the interactions of the two agonists. In our model, a hydrogen bond is formed between the indole hydrogen of 5-HT and the residue TYR153. This interaction is not present in the case of mCPBG. Conversely, a hydrogen bond exists between SER182 and a protonated nitrogen of mCPBG, which does not exist in 5-HT. Our modeling results were found to be in accordance with experimental data.     

25-Hydroxyvitamin D3 is an agonistic vitamin D receptor ligand

25-Hydroxyvitamin D₃ 1α-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D₃ into 1α,25-dihydroxyvitamin D₃, a vitamin D receptor ligand. 25-Hydroxyvitamin D₃ has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1α-hydroxylase-specific inhibitor we provide in four cellular systems, primary mouse kidney, skin, prostate cells and human MCF-7 breast cancer cells, evidence that 25-hydroxyvitamin D₃ has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D₃ internalisation, in Cyp27b1^?/? cells explains their higher sensitivity to 25-hydroxyvitamin D₃. 25-Hydroxyvitamin D₃ action depends on the vitamin D receptor signalling supported by the unresponsiveness of the vitamin D receptor knockout cells. Molecular dynamics simulations show the identical binding mode for both 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D₃. Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D₃ in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D₃ with 1α,25-dihydroxyvitamin D₃ in Cyp27b1^?/? cells further demonstrates the agonistic action of 25-hydroxyvitamin D₃ and suggests that a synergism between 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ might be physiologically important. In conclusion, 25-hydroxyvitamin D₃ is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties.     

Mechanism of primary and secondary ion-radical pair formation in photosystem I complexes

The mechanisms of the ultrafast charge separation in reaction centers of photosystem I (PS I) complexes are discussed. A kinetic model of the primary reactions in PS I complexes is presented. The model takes into account previously calculated values of redox potentials of cofactors, reorganization energies of the primary P700⁺A ₀ ^- and secondary P700⁺A ₁ ^- ion-radical pairs formation, and the possibility of electron transfer via both symmetric branches A and B of redox-cofactors. The model assumes that the primary electron acceptor A₀ in PS I is represented by a dimer of chlorophyll molecules Chl2A/Chl3A and Chl2B/Chl3B in branches A and B of the cofactors. The characteristic times of formation of P700⁺A ₀ ^- and P700⁺A ₁ ^- calculated on the basis of the model are close to the experimental values obtained by pump-probe femtosecond absorption spectroscopy. It is demonstrated that a small difference in the values of redox potentials between the primary electron acceptors A_0A and A_0B in branches A and B leads to asymmetry of the electron transfer in a ratio of 70: 30 in favor of branch A. The secondary charge separation is thermodynamically irreversible in the submicrosecond range and is accompanied by additional increase in asymmetry between the branches of cofactors of PS I.     

Mehler-peroxidase reaction mediates zeaxanthin formation and zeaxanthin-related fluorescence quenching in intact chloroplasts

Induction of zeaxanthin formation and the associated nonphotochemical quenching in iodoacetamide-treated, non-CO₂-fixing intact chloroplasts of Lactuca sativa L. cv Romaine is reported. The electron transport needed to generate the required ΔpH for zeaxanthin formation and nonphotochemical quenching are ascribed to the Mehler-ascorbate peroxidase reaction. KCN, an inhibitor of ascorbate peroxidase, significantly affected these activities without affecting linear electron transport to methyl viologen or violaxanthin deepoxidase activity. At 1 millimolar KCN, zeaxanthin formation and ΔpH were inhibited 60 and 55%, respectively, whereas ascorbate peroxidase activity was inhibited almost totally. The KCN-resistant activity, which apparently was due to electron transport mediated by the Mehler reaction alone, however, was insufficient to support a high level of nonphotochemical quenching. We suggest that in vivo, as CO₂ fixation becomes limiting, the Mehler-peroxidase reaction protects photosystem II against the excess light by supporting the electron transport needed for zeaxanthin-dependent nonphotochemical quenching and concomitantly scavenging H₂O₂. Ascorbate is essential for this process to occur.     

Activation of non-photochemical quenching in thylakoids and leaves

The mechanism of rapidly-relaxing non-photochemical quenching in two plant species,Chenopodium album L. andDigitalis purpurea L., that differ considerably in their capacity for such quenching has been investigated (Johnson G.N. et al. 1993, Plant Cell Environ.16, 673–679). Illumination of leaves of both species in the presence of 2% O₂ balance N₂ led to the formation of zeaxanthin. When thylakoids were isolated from leaves of each species that had been so treated it was found that inD. purpurea non-photochemical quenching was “activated” relative to the control; a higher level of quenching was found for a given trans-thylakoid pH gradient. No such activation of non-photochemical quenching was observed inC. album. Similar conclusions were drawn when comparing quenching in intact leaves. It is concluded that light activation of quenching is a process that cannot readily be induced inC. album. Measurement of the sensitivity of non-photochemical quenching in leaves ofC. album andD. purpurea to dithiothreitol (DTT; a reagent that inhibits formation of zeaxanthin) showed differences between the two species. In both cases, feeding leaves with DTT inhibited the light-induced formation of zeaxanthin. InC. album this was accompanied by complete inhibition of reversible non-photochemical quenching, whereas inD. purpurea this inhibition was only partial. Data are discussed in relation to studies on the mechanism of quenching and the role of zeaxanthin in this process.     

Activity of 25-Hydroxylase in Human Gingival Fibroblasts and Periodontal Ligament Cells

Background

We previously demonstrated that 25-hydroxyvitamin D₃ concentrations in gingival crevicular fluid are 300 times higher than those in the plasma of patients with aggressive periodontitis. Here we explored whether 25-hydroxyvitamin D₃ can be synthesized by periodontal soft tissue cells. We also investigated which of the two main kinds of hydroxylases, CYP27A1 and CYP2R1, is the key 25-hydroxylase in periodontal soft tissue cells.

Methodology/Principal Findings

Primary cultures of human gingival fibroblasts and periodontal ligament cells from 5 individual donors were established. CYP27A1 mRNA, CYP2R1 mRNA and CYP27A1 protein were detected in human gingival fibroblasts and periodontal ligament cells, whereas CYP2R1 protein was not. After incubation with the 25-hydroxylase substrate vitamin D₃, human gingival fibroblasts and periodontal ligament cells generated detectable 25-hydroxyvitamin D₃ that resulted in the production of 1α,25-dihydroxyvitamin D₃. Specific knockdown of CYP27A1 in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ production. Knockdown of CYP2R1 did not significantly influence 25-hydroxyvitamin D₃ synthesis. Sodium butyrate did not influence significantly CYP27A1 mRNA expression; however, interleukin-1β and Porphyromonas gingivalis lipopolysaccharide strongly induced CYP27A1 mRNA expression in human gingival fibroblasts and periodontal ligament cells.

Conclusions

The activity of 25-hydroxylase was verified in human gingival fibroblasts and periodontal ligament cells, and CYP27A1 was identified as the key 25-hydroxylase in these cells.     

Reactive column HPLC: rapid analysis on RPC-5 of oligoadenylates that differ at the 3'-terminus.

Oligoadenylates can be analyzed according to the type of 3′-terminus (A_nA, A_nAp, and A_nA > p, oligoadenylates that have at the 3′-terminus no phosphate, a 2′(3′)-monophosphate, and a 2′,3′-cyclic phosphate respectively) by hplc on RPC-5 support using a novel dual-column technique. The first column separates A_nAp plus A_nA > p from A_nA, and at the start of the second column a layer of bacterial alkaline phosphatase enzyme converts the A_nAp into A_nA. Hence this A_nA emerges separately from the original A_nA and from the A_nA > p. The technique can be used to analyze a three-component mixture for a single chain length or a mixture of A_nAp and A_nA > p of mixed chain lengths (n = 3 to 7). The presence of poly(U) does not interfere with the analysis.     

Metabolic and energetic aspects of biohydrogen production of Clostridium tyrobutyricum: The effects of hydraulic retention time and peptone addition

This study evaluates the microbial metabolism and energy demand in fermentative biohydrogen production using Clostridium tyrobutyricum FYa102 at different hydraulic retention times (HRT) over a period of 1-18 h. The hydrogen yield shows a positive correlation with the butyrate yield, the B/A ratio, and the Y_H2/2(Y_HAc+Y_HBu) ratio, but a negative correlation with the lactate yield. A decrease in HRT, which is accompanied by an increased biomass growth, tends to decrease the B/A ratio, due presumably to a higher energy demand for microbial growth. The production of lactate at a low HRT, however, may involve an unfavorable change in e⁻ equiv distribution to result in a reduced hydrogen production. Finally, the relatively high hydrogen yields observed in the bioreactor with the peptone addition may be ascribed to the utilization of peptone as an additional energy and/or amino-acid source, thus reducing the glucose demand for biomass growth during the hydrogen production process.     

Synthesis and pharmacological evaluation of novel 1,3,8- and 1,3,7,8-substituted xanthines as adenosine receptor antagonists

A number of novel xanthines bearing a variety of substituents at positions 1, 3, 7 and 8 were prepared and evaluated for their binding affinity to the human adenosine receptor A₁, A_2A, A_2B and A₃ subtypes. Several of the 1,3,8- and 1,3,7,8-substituted xanthines showed moderate-to-high affinity at human A_2B and A₁ receptors, with the most active compound (14q) having a pK_i of 7.57 nM for hA_2B receptors and a selectivity over hA_2A receptors of 8.1-fold and hA₁ receptors of 3.7-fold.     

Docking and SAR studies of calystegines: Binding orientation and influence on pharmacological chaperone effects for Gaucher’s disease

We report on the identification of the required configuration and binding orientation of nor-tropane alkaloid calystegines against β-glucocerebrosidase. Calystegine B₂ is a potent competitive inhibitor of human lysosomal β-glucocerebrosidase with K_i value of 3.3 μM. A molecular docking study revealed that calystegine B₂ had a favorable van der Waals interactions (Phe128, Trp179, and Phe246) and the hydrogen bonding (Glu235, Glu340, Asp127, Trp179, Asn234, Trp381 and Asn396) was similar to that of isofagomine. All calystegine isomers bound into the same active site as calystegine B₂ and the essential hydrogen bonds formed to Asp127, Glu235 and Glu340 were maintained. However, their binding orientations were obviously different. Calystegine A₃ bound to β-glucocerebrosidase with the same orientations as calystegine B₂ (Type 1), while calystegine B₃ and B₄ had different binding orientations (Type 2). It is noteworthy that Type 1 orientated calystegines B₂ and A₃ effectively stabilized β-glucocerebrosidase, and consequently increased intracellular β-glucocerebrosidase activities in N370S fibroblasts, while Type 2 orientated calystegines B₃ and B₄ could not keep the enzyme activity. These results clearly indicate that the binding orientations of calystegines are changed by the configuration of the hydroxyl groups on the nor-tropane ring and the suitable binding orientation is a requirement for achieving a strong affinity to β-glucocerebrosidase.     

Hydrogen bonding: further evidence for the cause of the color change of nitrosylpentaamminechromium (III) compounds. Crystal structures of [Cr(NO)(NH3)5](PF6)2 and [Cr(NO)(NH3)5]Cl(PF6)

The crystal structures of [Cr(NO)(NH₃)₅](PF₆)₂ (red) and [Cr(NO)(NH₃)₅]Cl(PF₆) (brown) have been determined. The [Cr(NO)(NH₃)₅]²⁺(A) complex cations in these compounds have a slightly distorted octahedral geometry with a strictly linear Cr-N-O arrangement (from symmetry). The short interatomic distances (2.399 Å × 4) between the O (nitrosyl) and H (ammonia in adjacent complex cations) atoms in A(PF₆)₂ indicate the existence of hydrogen bonds, while the interatomic distances (3.258 Å × 8) between those in ACl(PF₆) are much longer, and the hydrogen bonds should be weak in spite of the presence of the smaller counter anion of chloride ion in ACl(PF₆). Comparisons of the five crystal structures of A(PF₆)₂, ACl₂, ACl(ClO₄), ACl(PF₆), and A(ClO₄)₂ have led to the conclusion that the existence of the strong hydrogen bonds gives red crystals of A(PF₆)₂, while the absence of hydrogen bonds results in the formation of green crystals of A(ClO₄)₂ (O ? H, 3.595 Å × 2). The color change of the crystals (from red to green) with the change of outer sphere anions is attributed to the change of the strength of the hydrogen bonding between the complex cations.     

Zeaxanthin and the Induction and Relaxation Kinetics of the Dissipation of Excess Excitation Energy in Leaves in 2% O(2), 0% CO(2)

The relationship between the carotenoid zeaxanthin, formed by violaxanthin de-epoxidation, and nonphotochemical fluorescence quenching (q_NP) in the light was investigated in leaves of Glycine max during a transient from dark to light in 2% O₂, 0% CO₂ at 100 to 200 micromoles of photons per square meter per second. (a) Up to a q_NP (which can vary between 0 and 1) of about 0.7, the zeaxanthin content of leaves was linearly correlated with q_NP as well as with the rate constant for radiationless energy dissipation in the antenna chlorophyll (k_D). Beyond this point, at very high degrees of fluorescence quenching, only k_D was directly proportional to the zeaxanthin content. (b) The relationship between zeaxanthin and k_D was quantitatively similar for the rapidly relaxing quenching induced in 2% O₂, 0% CO₂ at 200 micromoles of photons per square meter per second and for the sustained quenching induced by long-term exposure of Nerium oleander to drought in high light (B Demmig, K Winter, A Krüger, F-C Czygan [1988] Plant Physiol 87: 17-24). These findings suggest that the same dissipation process may be induced by very different treatments and that this particular dissipation process can have widely different relaxation kinetics. (c) A rapid induction of strong nonphotochemical fluorescence quenching within about 1 minute was observed exclusively in leaves which already contained a background level of zeaxanthin.