Comparative study of the interaction mechanism of astilbin,isoastilbin, and neoastilbin with CYP3A4

The interactions of human CYP3A4 with three selected isomer flavonoids, such as astilbin, isoastilbin and neoastilbin, were clarified using spectral analysis, molecular docking, and molecular dynamics simulation. During binding with the three flavonoids, the intrinsic fluorescence of CYP3A4 was statically quenched in static mode with nonradiative energy conversion. The fluorescence and ultraviolet/visible (UV/vis) data revealed that the three flavonoids had a moderate and stronger binding affinity with CYP3A4 due to the order of the K_a1 and K_a2 values ranging from 10⁴ to 10⁵ L·mol⁻¹. In addition, astilbin had the highest affinity with CYP3A4, then isoastilbin and neoastilbin, at the three experimental temperatures. Multispectral analysis confirmed that binding of the three flavonoids resulted in clear changes in the secondary structure of CYP3A4. It was found from fluorescence, UV/vis and molecular docking analyses that these three flavonoids strongly bound to CYP3A4 by means of hydrogen bonds and van der Waals forces. The key amino acids around the binding site were also elucidated. Furthermore, the stabilities of the three CYP3A4 complexes were evaluated using molecular dynamics simulation.     

Effects of supplied cinnamic acids and biosynthetic intermediates on the anthocyanins accumulated by wild carrot suspension cultures

Anthocyanins isolated and characterized from the wild carrot suspension cultures used here were 3-O--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D<-galactopyranosylcyanidin (1), 3-O-[-D- xylopyranosyl-(12)--D-galactopyranosyl]cyanidin (2), 3-O-(6-O-sinapoyl)--D-glucopyranosyl-(16)-[-D- xylopyranosyl-(12)-]-D-galactopyranos ylcyanidin (3), 3-O-(6-O-feruoyl)--D-glucopyranosyl-(16)-[- D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (4), 3-O-(6-O-coumaroyl)--D-glucopyranosyl-(16)- [-D-xylopyranosyl-(12)-]-D-galactopyrano sylcyanidin (5), 3-O-[6-O-(3,4,5-trimethoxycinnamoyl)]-- D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (6), 3-O-[6-O-(3,4-dime- thoxycinnamoyl)]--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (7), 3-O-[(6-O-sinapoyl)--D-glucopyranosyl-(16)--D-galactopyranosyl]cyanidin (8), and 3-O-(-D-galactopyranosyl)cyanidin (9). Except when cinnamic acids were provided in the culture medium, the major anthocyanin present in the two clones examined was 2. When the naturally occurring and some non-naturally occurring cinnamic acids were provided individually in the medium, 1 and 2 were minor components and the anthocyanin acylated with the supplied cinnamic acid, namely 3, 4, 5, 6, or 7 was the major anthocyanin present in the tissue. When caffeic acid was provided the major anthocyanin in the tissue was 4, thereby suggesting that the caffeic acid was methylated before its use in anthocyanin biosynthesis. Other cinnamic acids supplied had limited effects on the anthocyanins accumulated and appeared not to result in the accumulation of new anthocyanins by the tissue. Thus the tissue can use some but not all analogues of sinapic acid to acylate anthocyanins. Additional anthocyanins were detected in extracts of the wild carrot tissue cultures using mass spectrometry (both MS/MS and HPLC/MS). The additional compounds detected have also been found in cultures of black carrot, an Afghan cultivar of Daucus carota ssp. sativa and the flowers of wild carrot giving no evidence for qualitative differences in the anthocyanins synthesized by subspecies, cell cultures from subspecies, or clones from cell cultures. There are major differences in the amounts of individual anthocyanins found in cultures from different subspecies and in different clones from cell cultures. Here anthocyanins without acyl groups were usually found in the tissues and their accumulation is discussed. On the basis of the structures of the isolated anthocyanins, a likely pathway from cyanidin to the accumulated anthocyanins is proposed and discussed.Abbreviations Sin sinapoyl - Fer feruoyl - 4-Coum. 4-coumaroyl - 3,4-MeO₂Cin 3,4-dimethoxyeinnamoyl - 3,4,5-MeO₃Cin 3,4,5-trimethoxycinnamoyl - Cya cyanidin     

Selective Expression of PNA-Binding Glycoconjugates by Invasive Human Melanomas: A New Marker of Metastatic Potential

Alterations of cell-surface glycoconjugates have been associated with invasiveness and metastatic capacity in a number of experimental and human tumors (bladder and colon cancer). We have recently shown that human melanoma cells from variants selected for high metastatic potential in an animal model bind the lectin peanut agglutinin (PNA), and that human melanoma cell populations enriched for PNA binding cells generate a higher frequency of metastases when xenografted into immune suppressed neonatal rats. We have therefore sought cells binding PNA in biopsied human melanocytic tumors and compared frequencies of PNA binding by cells from benign nevi, early and late primary melanomas, and metastatic melanomas. Sections of conventionally processed tissues were deparaffinised and exposed to biotinylated PNA; PNA fixation was revealed by the avidine/peroxidase/AEC technique. In 51 specimens tested, PNA appears to react electively with invasive tumors, since only one of the 7 early primary melanomas (Clark III) reacted while 13/23 late primary melanomas (Clark III-V), and 4/21 melanoma metastases were reactive. In addition, only 1/17 benign nevi bound PNA. In primary tumors, the reactive cells were exclusively invasive tumors cells in the dermis. PNA reactive material was observed in the cytoplasm and plasma membrane of reactive cells. Hence, alterations in composition and cellular localisation of glycoconjugates detectable by lectin histochemistry in melanoma cells may be markers of metastatic potential that may be applicable on an individual patient basis.     

Genetic control of UDPglucose:flavonol 3-O-glucosyltransferase in the endosperm of maize

The enzyme UDPglucose:flavonol 3-O-glucosyltransferase is shown to be under the coordinate control of three genes involved in anthocyanin biosynthesis in the aleurone of maize: C, R, and Bz. Of the three, Bz appears to be the structural gene. Data presented here (dosage comparisons, induction in the mutant c-p, and effect of paramutation at R) indicate that the enzyme is inducible by substances resulting from the action of the C and R genes and that active forms of C and R are required for this induction. Mechanisms of regulation of the Bz gene by C and R are discussed.Laboratory of Genetics Paper No. 2032. Research support by the College of Agricultural and Life Sciences and by National Institutes of Health Grant No. 15422.     

Sulfated glycoproteins and extracellular matrix of cultured human pulmonary endothelial cells

Endothelial cells derived from human pulmonary arteries incorporate (³H)-glucosamine and ³⁵SO₄ into glycosaminoglycans and into the carbohydrate side chains of glycoproteins. These ³H/³⁵S-carbohydrate chains were isolated from cells and culture medium after Pronase digestion. The ³H/³⁵S-glycosaminoglycans were separated from the ³H/³⁵S glycopeptides by chromatography on Sephadex G-50. The distribution of cellular glycosaminoglycans and glycopeptides indicated that 30–60% of the cellular ³⁵S-glycopeptides may be associated with the matrix components that are synthesized by the cell and attached to a plastic substratum. Human pulmonary arterial endothelial cells were grown on collagen or on a matrix derived from vascular smooth muscle cells in order to investigate how smooth muscle cell extracellular matrix components may regulate the synthesis of endothelial cell glycoconjugates. Endothelial cells grown on plastic release various proportions of the glycoconjugates they synthesize into the culture medium. However, these same cells, when grown on substratum composed of extracellular matrix materials, synthesized altered proportions of cell-associated glycosaminoglycans and reduced the levels of total glycosaminoglycans they released into the culture medium. Thus the growth of endothelial cells on a matrix of smooth muscle cell components indicates that the glycosaminoglycan materials released into the culture medium by cells grown on a plastic substratum may not be an accurate reflection of the levels or composition of extracellular matrix materials made by endothelial cells in vivo.     

Flavonoid patterns in leaves of the gramineae

In a leaf survey of 274 species from 121 genera of the Gramineae, flavone C-glycosides and tricin were found to be the major flavonoids in 93% of the samples. By contrast, apigenin and luteolin O-glycosides were comparatively rare, as were the flavonols, kaempferol and quercetin. In only one species, Rottboellia exaltata were flavonols the sole flavonoids. 7.3′.4′-Trihydroxyflavone, which has been detected in the Juncaceae, was found in 3 of 5 samples of the species Bothriochloa bladhii. Flavonoid sulphates were present in 16% of the species examined. While in most of these plants tricin glycosides were conjugated with sulphate, in Paspalum convexum quercetin mono- and di-sulphates and 1-caffeylglucose sulphate were identified. Flavonoid sulphates are present in the tropical-subtropical subfamilies: Panicoideae (in 18% of species). Chloridoideae (15%) and Arundinoideae (40%) but were not found at all in tribes of the cool temperate regions. Proanthocyanidins were found in only 3% of the species surveyed. The flavan-4-ol, luteoforol and its apigenin analogue were detected only in the subfamilies Panicoideae and Chloridoideae, where they occured in 12 and 6% of species respectively.     

The carbohydrate deposits detected by histochemical methods in the molecular layer of the dentate gyrus in the hippocampal formation of patients with schizophrenia, Down's syndrome and dementia, and aged person

Post-mortem brain tissue was obtained from 28 patients with brain disorders, of which 15 had clinically diagnosed schizophrenia, 6 Alzheimer type dementia, 5 dementia with tangles and 2 cases of Down's syndrome. The controls were 22 cases from autopsies without brain disorders or with no known episodes of brain disorder. The tissues were stained for the detection of carbohydrate deposits in the hippocampal formation, using lectin, immunohistochemical and conventional staining methods. The staining revealed the existence of spherical deposits in the inner and middle molecular layers of the dentate gyrus in the hippocampal formation which contained fucose, galactose, N-acetyl galactosamine, N-acetyl glucosamine, sialic acid, mannose and chondroitin sulfate. The number of the deposits was higher in patients with brain disorder such as schizophrenia, Alzheimer type dementia, dementia with tangles or Down's syndrome, and in some aged individuals, in comparison to those in younger individuals. No deposits were detected in a few younger or aged individuals. Spherical deposits 3–10[emsp4 ]m in diameter may be an immature form of the corpora amylacea, since they were similar in the histochemical characteristics with lectin, immunohistochemical and conventional staining methods. However, differing staining ability by hematoxylin, periodic acid Schiff's reagent and antibodies against the intracellular degraded proteins such as ubiquitin and tau-protein was observed. The antibodies against ubiquitin and tau-protein showed clear reactivity with the corpora amylacea and no reactivity with spherical deposits, indicating that the corpora amylacea has an intracellular origin and spherical deposits an extracellular matrix origin. The results obtained in this study indicate that not only neuronal degeneration but also unusual glycometabolism in neurons may disturb the neuronal function and cause brain disorders, and that spherical deposits may cause dysfunction of the neuronal network in the dentate gyrus of the hippocampus which is closely linked with recognition and memory functions.     

High molecular weight neoglycoconjugates for solid phase assays

Adsorption of a carbohydrate on solid phase is the necessary stage of the immunosorbent assay (ELISA) and analogous methods of the study of carbohydrate-protein interaction. Usually physical adsorption on polystyrene requires a high concentration of conjugated carbohydrate and, thus, enormous consumption of it. In this study, we explored two approaches allowing more rational use of oligosaccharide (Glyc). The first of them is based on the covalent immobilization of neoglycoconjugates on the NH₂-modified polystyrene; the second one is based on the elevated adherence of high m.w. neoglycoconjugates to polystyrene. Covalent immobilization of polyacrylamide conjugates, Glyc-PAA, provided a possibility to solve the problem, but the nonspecific binding of antibodies in ELISA proved to be unacceptably high. At the same time, the increase of the Glyc-PAA m.w. from 30 kDa to 2,000 kDa allowed a 10–20 fold decrease of its consumption, when using physical adsorption, whereas the assay background remained at the low level. The amount of 2,000 kDa Glyc-PAA that is sufficient for the coating of a standard 96-well plate corresponds to the nanomole level of oligosaccharide, this providing a possibility to use saccharides that are available in a very limited amount when studying the carbohydrate-protein interaction with solid-phase techniques. Published in 2005.     

Computational study of conformational and chiroptical properties of (2R,3S,4R)-(+)-3,3',4,4',7-flavanpentol

Conformational analysis of (2R,3S,4R)-(+)-3,3',4,4',7-flavanpentol, a flavonoid compound displaying both antioxidant and pro-oxidant properties, is performed by molecular mechanics and density functional theory calculations both in the gas phase and in methanol solution by using the Polarizable Continuum Model. Nine different conformations are identified. Absorption (UV) and circular dichroism (CD) spectra and optical rotations are calculated by means of time dependent density functional theory (TDDFT) and compared with experiments. The effects of a complex environment formed by water and proline-rich peptide molecules on the conformational characteristics of (2R,3S,4R)-(+)-3,3',4,4',7-flavanpentol and therefore on its UV and CD spectra are investigated by atomistic molecular dynamics simulations.     

Human plasma trans-sialidase donor and acceptor specificity

Earlier we have isolated from human plasma desialylated low density lipoproteins (dLDL) and showed that, first, dLDL induce cholesterol esters accumulation—the main process accompanying atherosclerosis development. Second, the process of lipoprotein desialylation took place in plasma, and, finally, sialic acids removed from LDL are transferred to other serum glycoconjugates. In this study we have isolated from human plasma an enzyme transferring sialic acid residues (trans-sialidase) by affinity chromatography and studied its donor and acceptor specificity. Isolated enzyme in the presence of saccharide acceptor can remove sialic acids from different lipoproteins, glycoproteins (fetuin, transferrin), and gangliosides (GM3, GD3, GM1, GD1a, GD1b). Plasma enzyme translocates 2-6, 2-3 and to a lower extent 2-8 bonded sialic acids. Sialoglycoconjugates of human serum erythrocytes, serum lipoproteins, glycoproteins, and gangliosides can serve as donors of sialic acid for trans-sialidase. Desialylated lipoproteins, especially dLDL,are more preferable sialic acid acceptors. Transferred sialic acid is found to be 2-6, 2-3,and 2-8 connected.