Oblongionosides A–F,megastigmane glycosides from the leaves of Croton oblongifolius Roxburgh

From the 1-BuOH-soluble fraction of a MeOH extract of the leaves of Croton oblongifolius Roxburgh, collected in Chiang Mai, Thailand, six megastigmane glycosides, named oblongionosides A–F were isolated together with eight known compounds, and their structures elucidated on the basis of spectroscopic data. Absolute structures were determined by HPLC analyses and application of the modified Mosher’s method.     

A computational tool to predict the evolutionarily conserved protein–protein interaction hot-spot residues from the structure of the unbound protein

Identifying hot-spot residues – residues that are critical to protein–protein binding – can help to elucidate a protein’s function and assist in designing therapeutic molecules to target those residues. We present a novel computational tool, termed spatial-interaction-map (SIM), to predict the hot-spot residues of an evolutionarily conserved protein–protein interaction from the structure of an unbound protein alone. SIM can predict the protein hot-spot residues with an accuracy of 36–57%. Thus, the SIM tool can be used to predict the yet unknown hot-spot residues for many proteins for which the structure of the protein–protein complexes are not available, thereby providing a clue to their functions and an opportunity to design therapeutic molecules to target these proteins.     

Synthesis of haem cytochrome c prosthetic group from δ-aminolaevulinate by the cell sap from rat liver

To determine whether the prosthetic group of cytochrome c is synthesized and linked to the apoprotein in the cytosol or in connexion with the endoplasmic reticulum, we have studied the incorporation in vitro of delta-amino[(14)C]laevulinate into porphyrin compounds and cytochrome c by the cell sap from rat liver. The radioactive precursor was incorporated into a trichloroacetic acid-precipitable form partially resistant to extractions by acid solvents, suggesting the existence of a fraction covalently linked to protein. The activity was proportional to the amount of protein incubated, did not increase substantially by supplementation with the microsomal fraction and an energy source, and was very low in the pH5 fraction. Addition of increasing amounts of haemin inhibited the incorporation, as with purified delta-aminolaevulinate dehydratase. [(14)C]Protoporphyrin IX was identified by paper chromatography, together with a shoulder running as protohaem IX. The cell sap in the absence of ribosomes was also able to incorporate radioactivity into purified cytochrome c, and the addition of ribosomes significantly enhanced the activity. The precursors of haem c were synthesized in the soluble system by the known haem-synthetic pathway, as shown by the kinetics of labelling of the coproporphyrin, protoporphyrin and haem fractions, and the activities were concentrated in the precipitate obtained between 40 and 60% saturation with (NH(4))(2)SO(4). The presence of ferrochelatase was indicated by the incorporation of (55)Fe into proto- and haemato-haem identified by paper chromatography. It is concluded that the cell sap from rat liver contains the complete set of enzymes for the synthesis from delta-aminolaevulinate of haem c and its linkage to a small pool of free apoprotein c present in soluble form. This suggests that an ancillary pathway of haem synthesis occurs in the cytosol for at least the formation of the prosthetic group, which is linked post-translationally to that pool of apoprotein c synthesized by free polyribosomes.     

Flash-induced reduction of cytochrome b-559 by Q infB sup− in chloroplasts in the presence of protonophores

Flash-induced, fast (t _1/2 1 ms), reversible reduction of the high potential cytochrome b-559 (cyt b-559HP) was observed in chloroplasts in the presence of 2 M protonophore, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), CCCP (carbonylcyanide 3-chlorophenylhydrazone) or SF 6847 (2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)phenol). These protonophores promote autooxidation of cyt b-559HP in the dark (Arnon and Tang 1988, Proc Natl Acad Sci USA 85: 9524). No fast photoreduction could, however, be observed if the molecules were oxidized with ferricyanide in the absence of protonophores. This suggests that the molecules must be deprotonated to be capable for fast photoreduction.Photoreduction of cyt b-559HP was largely insensitive to DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), but was inhibited by DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). With a train of flashes, no oscillation could be observed in the amplitudes of photoreduction. These data strongly suggest that cyt b-559HP is reduced by the semireduced secondary quinone acceptor (Q_B ^–) of Photosystem 2.Abbreviations ADRY- acceleration of the deactivation reactions of the water-splitting enzyme system Y of photosynthesis - Ant 2p- 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - cyt- cyto-chrome - CCCP- carbonylcyanide 3-chlorophenylhydrazone - DBMIB- 2,5-dibromo-3-methyl-6-iso-propyl-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimehtylurea - FCCP- carbonylcyanide p-trifluoromethoxyphenylhydrazone - FeCy- ferricyanide - HP- high potential form - HQ- hydroquinone - PQ- plastoquinone - PS 2- Photosystem 2 - SF 6847- 2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)-phenol     

On the decarboxylation of α-keto acids by isolated chloroplasts

The mechanism of the decarboxylation of α-keto acids by isolated chloroplasts has been studied with the aid of superoxide dismutase and catalase. Using photosynthetic and enzymatic systems, which are known to catalyze peroxidic oxidations, we have been able to demonstrate that both the superoxide free radical ion and H₂O₂ are necessary for maximal rates of decarboxylation. In isolated chloroplasts, an auto-oxidizable electron acceptor as well as an electron donor for Photosystem I are absolute requirements for the decarboxylation. H₂O₂ seems to be the primary oxidant in the decarboxylation of pyruvate or glyoxylate by isolated chloroplasts. A secondary rate of decarboxylation is superimposed on the primary one, mediated by superoxide free radical ion. Mn²⁺ stimulates the decarboxylation probably via intermediarily-formed Mn³⁺ in a reaction, which is neither inhibited by catalase nor by superoxide dismutase. A decarboxylation of pyruvate or glyoxylate by isolated chloroplasts in the presence of NADP⁺ is initiated, as soon as the available NADP⁺ is fully reduced. In this case, the open-chain electron transport seems to switch from NADP⁺ to oxygen as the terminal electron acceptor.     

Microtropiosides A–F: ent-Labdane diterpenoid glucosides from the leaves of Microtropis japonica (Celastraceae)

From a 1-BuOH-soluble fraction of a MeOH extract of the leaves of Microtropis japonica, collected in the Okinawa islands, six ent-labdane glucosides, named microtropiosides A–F, were isolated together with one known acyclic sesquiterpene glucoside. Their structures were elucidated by a combination of spectroscopic analyses, and their absolute configurations determined by application of the β-d-glucopyranosylation-induced shift-trend rule in ¹³C NMR spectroscopy and the modified Mosher’s method.     

Oxidation of phenolic compounds by the bifunctional catalase–phenol oxidase (CATPO) from Scytalidium thermophilum

The thermophilic fungus Scytalidium thermophilum produces a novel bifunctional catalase with an additional phenol oxidase activity (CATPO); however, its phenol oxidation spectrum is not known. Here, 14 phenolic compounds were selected as substrates, among which (+)-catechin, catechol, caffeic acid, and chlorogenic acid yielded distinct oxidation products examined by reversed-phase HPLC chromatography method. Characterization of the products by LC-ESI/MS and UV–vis spectroscopy suggests the formation of dimers of dehydrocatechin type B (hydrophilic) and type A (hydrophobic), as well as oligomers, namely, a trimer and tetramer from (+)-catechin, the formation of a dimer and oligomer of catechol, a dimer from caffeic acid with a caffeicin-like structure, as well as trimeric and tetrameric derivatives, and a single major product from chlorogenic acid suggested to be a dimer. Based on the results, CATPO oxidizes phenolic compounds ranging from simple phenols to polyphenols but all having an ortho-diphenolic structure in common. The enzyme also appears to have stereoselectivity due to the oxidation of (+)-catechin, but not that of epicatechin. It is suggested that CATPO may contribute to the antioxidant mechanism of the fungus and may be of value for future food and biotechnology applications where such a bifunctional activity would be desirable.     

Improvement of the efficiency of transglycosylation catalyzed by α-galactosidase from Thermotoga maritima by protein engineering

At high concentrations of p-nitrophenyl-α-D-galactopyranoside (pNPGal) as a substrate, its hydrolysis catalyzed by α-galactosidase from Thermotoga maritima (TmGalA) is accompanied by transglycosylation resulting in production of a mixture of (α1,2)-, (α1,3)-, and (α1,6)-p-nitrophenyl (pNP)-digalactosides. Molecular modeling of the reaction stage preceding the formation of the pNP-digalactosides within the active site of the enzyme revealed amino acid residues which modification was expected to increase the efficiency of transglycosylation. Upon the site-directed mutagenesis to the predicted substitutions of the amino acid residues, genes encoding the wild type TmGalA and its mutants were expressed in E. coli, and the corresponding enzymes were isolated and tested for the presence of the transglycosylating activity in synthesis of different pNP-digalactosides. Three mutants, F328A, P402D, and G385L, were shown to markedly increase the total transglycosylation as compared to the wild type enzyme. Moreover, the F328A mutant displayed an ability to produce a regio-isomer with the (α1,2)-bond at yield 16-times higher than the wild type TmGalA.     

Resveratrol protects rats from Aβ-induced neurotoxicity by the reduction of iNOS expression and lipid peroxidation

Alzheimer disease (AD) is an age-dependent neurodegenerative disease characterized by the formation of β-amyloid (Aβ)-containing senile plaque. The disease could be induced by the administration of Aβ peptide, which was also known to upregulate inducible nitric oxide synthase (iNOS) and stimulate neuronal apoptosis. The present study is aimed to elucidate the cellular effect of resveratrol, a natural phytoestrogen with neuroprotective activities, on Aβ-induced hippocampal neuron loss and memory impairment. On adult Sprague-Dawley rats, we found the injection of Aβ could result in a significant impairment in spatial memory, a marked increase in the cellular level of iNOS and lipid peroxidation, and an apparent decrease in the expression of heme oxygenase-1 (HO-1). By combining the treatment with Aβ, resveratrol was able to confer a significant improvement in spatial memory, and protect animals from Aβ-induced neurotoxicity. These neurological protection effects of resveratrol were associated with a reduction in the cellular levels of iNOS and lipid peroxidation and an increase in the production of HO-1. Moreover, the similar neurological and cellular response were also observed when Aβ treatment was combined with the administration of a NOS inhibitor, N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME). These findings strongly implicate that iNOS is involved in the Aβ-induced lipid peroxidation and HO-1 downregulation, and resveratrol protects animals from Aβ-induced neurotoxicity by suppressing iNOS production.     

Structural basis of protein–protein interaction studied by NMR

This paper describes efforts of the structural genomics project in the nuclear magnetic resonance (NMR) laboratory at the University of Science and Technology of China. This structural genomics project is biological-functional driven. Targets are mainly selected from two systems: proteins related with regulation of gene expression in humans and other eukaryotes, and proteins existing in the cell junction in humans. The majority of proteins selected from these two systems are related with human health and diseases, and some are potential drug targets. Twenty-five protein structures from Homo sapiens and other eukaryotes have been determined during last 5 years in this laboratory. Nuclear magnetic resonance (NMR) spectroscopy is highly suited to investigate molecular interactions at a close physiological condition and is particularly suited for the study of low-affinity, transient complexes. It can provide information on protein surface interaction, their complex structure, and their dynamic properties during protein recognition. Several examples are given in this paper.     

Alternative modulation of protein–protein interactions by small molecules

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Ficuschlorins A - D, lactone Chlorins from the leaves of ficus microcarpa

Four new lactone chlorins, ficuschlorins A – D ( 1 – 4 , resp.), and six known pheophytins were isolated from the leaves of Ficus microcarpa. The structures of these compounds were determined by 1D‐ and 2D‐NMR spectroscopy, and other techniques. New natural pheophytins were rarely obtained. In the past ten years, only three new pheophytins were isolated from natural sources.     

A structure–activity relationship study on antiosteoclastogenesis effect of triterpenoids from the leaves of loquat (Eriobotrya japonica)

Our previous results elucidated that the leaves of Eriobotrya japonica possessed the potential to suppress ovariectomy-induced bone mineral density deterioration, and ursolic acid, the major bioactive component in these leaves, suppressed the osteoclast differentiation. The aim of this study was to discover more candidates for development of novel antiosteoclastogenesis agents from the leaves of E. japonica. Phytochemical analysis following a cell-based osteoclastic tartrate-resistant acid phosphatase (TRAP) activity assay revealed 11 more compounds with a potent antiosteoclastogenesis effect. The potency of ursane-type triterpenoids from the leaves of E. japonica prompted us to investigate the structure–activity relationships underlying their antiosteoclastogenesis. The results revealed that both the hydroxyl group at C-3 and the carboxylic group at C-17 played indispensable roles in the antiosteoclastogenesis activity of ursane-type triterpenoids. The configuration at C-3 (a beta-form of the hydroxyl group) was found to be important for this activity. While introducing a hydroxyl group at C-19 increased the inhibitory activity of ursane-type triterpenoids carrying an alpha-form hydroxyl group at C-3. The bioactivity analyses of ursolic acid and oleanolic acid demonstrated that the antiosteoclastogenesis effect of ursolic acid may be related to different positions of the C-29 and C-30 methyl groups on the E-ring, since oleanolic acid showed limited activity. The addition of a hydroxyl group at C-2 would dramatically improve the inhibition of oleanane-type triterpenoids. Collectively, these findings could provide important clues for the improvement of multi-targeted antiosteoclastogenesis agents from the leaves of E. japonica.     

A guanosine 5′-triphosphate-dependent protein kinase is localized in the outer envelope membrane of pea chloroplasts

A guanosine 5-triphosphate (GTP)-dependent protein kinase was detected in preparations of outer chloroplast envelope membranes of pea (Pisum sativum L.) chloroplasts. The protein-kinase activity was capable of phosphorylating several envelope-membrane proteins. The major phosphorylated products were 23- and 32.5-kilo-dalton proteins of the outer envelope membrane. Several other envelope proteins were labeled to a lesser extent. Following acid hydrolysis of the labeled proteins, most of the label was detected as phosphoserine with only minor amounts detected as phosphothreonine. Several criteria were used to distinguish the GTP-dependent protein kinase from an ATP-dependent kinase also present in the outer envelope membrane. The ATP-dependent kinase phosphorylated a very different set of envelope-membrane proteins. Heparin inhibited the GTP-dependent kinase but had little effect upon the ATP-dependent enzyme. The GTP-dependent enzyme accepted phosvitin as an external protein substrate whereas the ATP-dependent enzyme did not. The outer membrane of the chloroplast envelope also contained a phosphotransferase capable of transferring labeled phosphate from [-³²P]GTP to ADP to yield (-³²P]ATP. Consequently, addition of ADP to a GTP-dependent protein-kinase assay resulted in a switch in the pattern of labeled products from that seen with GTP to that typically seen with ATP.Abbreviations GDP (GMP, GTP) guanosine 5-diphosphate (mono-, tri-); kDa-kilodalton - S_0.5 concentration of substrate supporting half-maximal velocity - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tricine N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine     

α-Glucosidase-inhibitory iminosugars from the leaves of Suregada glomerulata

A water extract of the leaves of Suregada glomerulata (Euphorbiaceae) was found to inhibit rat small intestinal α-glucosidase. An examination of the extract afforded 20 iminosugars including one pyrrolidine and 19 piperidines. The structures of the 10 new compounds (11–20) were determined by NMR, and MS spectroscopic data analyses, and chemical correlations. The novelty of the identified compounds mainly stems from the loss of a hydroxy at C-4 and the presence of an 8-hydroxyoctyl side chain. Nine N-alkyl derivatives including N-methyl (1a, 8a, and 13a), N-butyl (1b, 2b, and 9b) and N,N-dimethyl (1c, 2c, and 9c) were synthesized. The compounds were tested for rat small intestinal α-glucosidase inhibitory activity. In total, 15 compounds, including compounds 11, 12, 15, and 19 and the three derivatives 8a, 9b, and 13a, showed inhibitory activity with IC₅₀ values less than 40 μM. In vivo results showed that total alkaloids of S. glomerulata (10 mg/kg) and four major iminosugars 1, 2, 3, and 9 (10 mg/kg) can lower the postprandial blood glucose level after sucrose and starch load in healthy male ICR mice.     

Prenylated C6–C3 compounds from the roots of Illicium henryi

Eleven prenylated C₆–C₃ compounds, illihenryifunones A, B (1, 2), illihenryifunol A (3), illihenryipyranol A (4), illihenryiones A−G (5–11), and three known prenylated C₆–C₃ compounds (12–14), were isolated from the roots of Illicium henryi. Structures of 1–11 were elucidated by spectroscopic methods including NMR, HRESIMS, and CD. The absolute configuration of the 11,12-diol moiety in 5 was determined by observing its induced circular dichroism after addition of Mo₂(OAc)₄ in DMSO. The absolute configuration of C-11 in 4 was determined as S based on the Rh₂(OCOCF₃)₄-induced CD data; the absolute configuration of 3 was determined as R by comparison of its experimental and calculated electronic circular dichroism (ECD). The antioxidant activities of compounds 1–14 were also evaluated. Compound 4 exhibited strong antioxidant activity with an IC₅₀ value of 2.97 ± 1.30 μM, whereas compounds 3 and 8 showed antioxidant activities with IC₅₀ values of 44.36 ± 0.30 and 48.00 ± 2.01 μM, respectively.     

The Rieske Fe/S protein of the cytochrome b6/f complex in chloroplasts: missing link in the evolution of protein transport pathways in chloroplasts?

The Rieske Fe/S protein, a nuclear-encoded subunit of the cytochrome b(6)/f complex in chloroplasts, is retarded in the stromal space after import into the chloroplast and only slowly translocated further into the thylakoid membrane system. As shown by the sensitivity to nigericin and to specific competitor proteins, thylakoid transport takes place by the DeltapH-dependent TAT pathway. The Rieske protein is an untypical TAT substrate, however. It is only the second integral membrane protein shown to utilize this pathway, and it is the first authentic substrate without a cleavable signal peptide. Transport is instead mediated by the NH(2)-terminal membrane anchor, which lacks, however, the twin-arginine motif indicative of DeltapH/TAT-dependent transport signals. Furthermore, transport is affected by sodium azide as well as by competitor proteins for the Sec pathway in chloroplasts, demonstrating for the first time some cross-talk of the two pathways. This might take place in the stroma where the Rieske protein accumulates after import in several complexes of high molecular mass, among which the cpn60 complex is the most prominent. These untypical features suggest that the Rieske protein represents an intermediate or early state in the evolution of the thylakoidal protein transport pathways.     

Electron-paramagnetic-resonance studies of the mechanism of leaf nitrite reductase. Signals from the iron–sulphur centre and haem under turnover conditions

Low-temperature e.p.r. spectra are presented of nitrite reductase purified from leaves of vegetable marrow (Cucurbita pepo). The oxidized enzyme showed a spectrum at g=6.86, 4.98 and 1.95 corresponding to high-spin Fe(3+) in sirohaem, which disappeared slowly on treatment with nitrite. The midpoint potential of the sirohaem was estimated to be -120mV. On reduction with Na(2)S(2)O(4) or Na(2)S(2)O(4)+Methyl Viologen a spectrum at g=2.038, 1.944 and 1.922 was observed, due to a reduced iron-sulphur centre. The midpoint potential of this centre was very low, about -570mV at pH8.1, decreasing with increasing pH. On addition of cyanide, which binds to haem, and Na(2)S(2)O(4), the iron-sulphur centre became further reduced. We think that this is due to an increased midpoint potential of the iron-sulphur centre. Other ligands to haem, such as CO and the reaction product NH(3), had similar but less pronounced effects, and also changed the lineshape of the iron-sulphur signal. Samples were prepared of the enzyme frozen during the reaction with nitrite, Methyl Viologen and Na(2)S(2)O(4) in various proportions. Signals were interpreted as due to the reduced iron-sulphur centre (with slightly different g values), a haem-NO complex and reduced Methyl Viologen. In the presence of an excess of nitrite, the haem-NO spectrum was more intense, whereas in the presence of an excess of Na(2)S(2)O(4) it was weaker, and disappeared at the end of the reaction. A reaction sequence is proposed for the enzyme, in which the haem-NO complex is an intermediate, followed by other e.p.r.-silent states, leading to the production of NH(4) (+).