Induction of G1 arrest and apoptosis in human jurkat T cells by pentagalloylglucose through inhibiting proteasome activity and elevating p27Kip1, p21Cip1/WAF1, and Bax proteins

Pentagalloylglucose, which is found in many medicinal plants, can arrest the cell cycle at G(1) phase through down-regulation of cyclin-dependent kinases 2 and 4 and up-regulation of the cyclin-dependent kinase inhibitors p27(Kip1) and p21(Cip1/WAF1) in human breast cancer cells. Pentagalloylglucose also induces apoptosis in human leukemic cells. However, the mechanisms by which pentagalloylglucose induces these effects is unclear. We now show that pentagalloylglucose inhibits the activities of purified 20 and 26 S proteasomes in vitro, the 26 S proteasome in Jurkat T cell lysates, and chymotrypsin-like activity of the 26 S proteasome in intact Jurkat T cells. The turnover of p27(Kip1) and p21(Cip1/WAF1), which is necessary for cell cycle progression mediated by proteasome degradation, was disrupted by treatment of human Jurkat T cells with pentagalloylglucose. This was shown by cycloheximide treatment and in vivo pulse-chase labeling experiments, and this effect correlated with the arrest of proliferation of Jurkat T cells at G(1). Inhibition of the proteasome by pentagalloylglucose and by the proteasome inhibitor MG132 caused accumulation of ubiquitin-tagged proteins in Jurkat T cells. The addition of pentagalloylglucose to Jurkat T cells enhanced the stability of the proteasome substrate Bax and increased cytochrome c release and apoptosis. Our findings suggest a mechanism for the effect of pentagalloylglucose on the cell cycle in human leukemic cells: that pentagalloylglucose down-regulates proteasome-mediated pathways because it is a proteasome inhibitor.     

Activation of the alternative pathway by pneumococcal cell walls.

The present studies were performed in order to identify the pneumococcal subcellular component responsible for activating the alternative pathway. Purified pneumococcal cell walls were able to activate the alternative pathway at a concentration as low as 5 mug/ml and were more active than crude cell walls, which in turn were more active than the whole organism. Purified pneumococcal cell membranes also were able to activiate the alternative pathway but had less than 10% of the activity of the purified walls. Thus, the cell wall appears to play a major role in pneumococcal activation of the alternative pathway. Pneumococcal cell walls containing ethanolamine were as effective as cell walls containing choline in activating the alternative pathway. Since C-reactive protein binds specifically to the phosphorylcholine residue of pneumococcal C-polysaccharide, it is unlikely that pneumococcal cell walls must combine with C-reactive protein in order to activate the alternative pathway.     

Recognition characters in peptide–polyphenol complex formation

Dietary polyphenols have received attention for their anti-oxidative, anti-carcinogenic and anti-neurodegenerative effects. Polyphenols bind to proteins leading to the formation of soluble or insoluble protein–polyphenol complexes which could significantly influence their biological activities. NMR and molecular modeling studies were performed to investigate the influence of the bulk, flexibility and hydrophobicity of polyphenols on the association with bradykinin, the peptide model. Our results show that the strength of the interactions could be positively correlated with polyphenol hydrophobicity and a comparison between pentagalloylglucose and vescalagin indicated that flexibility might play a positive role in the interaction with peptides and proteins.     

Recognition characters in peptide-polyphenol complex formation

Dietary polyphenols have received attention for their anti-oxidative, anti-carcinogenic and anti-neurodegenerative effects. Polyphenols bind to proteins leading to the formation of soluble or insoluble protein-polyphenol complexes which could significantly influence their biological activities. NMR and molecular modeling studies were performed to investigate the influence of the bulk, flexibility and hydrophobicity of polyphenols on the association with bradykinin, the peptide model. Our results show that the strength of the interactions could be positively correlated with polyphenol hydrophobicity and a comparison between pentagalloylglucose and vescalagin indicated that flexibility might play a positive role in the interaction with peptides and proteins.     

Subcellular localization of silicon and germanium in grass root and leaf tissues by SIMS: evidence for differential and active transport

Silicon transport and incorporation into plant tissue is important to both plant physiological function and to the influence plants have on ecosystem silica cycling. However, the mechanisms controlling this transport have only begun to be explored. In this study, we used secondary ion mass spectrometry (SIMS) to image concentrations of Si in root and shoot tissues of annual blue grass (Poa annua L.) and orchard grass (Dactylis glomerata L.) with the goal of identifying control points in the plant silica uptake pathway. In addition, we used SIMS to describe the distributions of germanium (Ge); the element used to trace Si in biogeochemical studies. Within root tissue, Si and Ge were localized in the suberized thick-walled region of endodermal cells, i.e. the proximal side of endodermal cells which is in close association to the casparian strip. In leaves, Si was present in the cell walls, but Ge was barely detectable. The selective localization of Si and Ge in the proximal side of endodermal cell walls of roots suggests transport control is exerted upon Si and Ge by the plant. The absence of Si in most root cell walls and its presence in the cell walls of leaves (in areas outside of the transpiration terminus) suggests modifications in the chemical form of Si to a form that favors Si complexation in the cell walls of leaf tissue. The low abundance of Ge in leaf tissue is consistent with previous studies that suggest preferential transport of Si relative to Ge.     

Atomic force microscopy of microfibrils in primary cell walls

Examination of angiosperm primary cell walls by transmission electron microscopy shows that they contain microfibrils that probably consist of cellulose microfibrils surrounded by associated non-cellulosic polysaccharides. Previous studies using solid-state (13)C NMR spectroscopy have shown that the cellulose is all crystalline with crystallites of cross-sectional dimensions of 2-3 nm. However, it is not known if each microfibril contains only one, or more than one crystallite because there is no agreement about the dimensions of the microfibrils. Partially hydrated primary cell walls isolated from onion ( Allium cepa L.) and Arabidopsis thaliana (L.) Heynh. were examined by atomic force microscopy and the microfibril diameters determined. The cell walls of both species contained tightly interwoven microfibrils of uniform diameter: 4.4+/-0.13 nm in the onion and 5.8+/-0.17 nm in A. thaliana. The effect was also examined of extracting the A. thaliana cell walls to remove pectic polysaccharides. The microfibrils in the extracted cell walls of A. thaliana were significantly narrower (3.2+/-0.13 nm) than those in untreated walls. The results are consistent with the microfibrils containing only one cellulose crystallite.     

Lignin isolated from primary walls of hybrid aspen cell cultures indicates significant differences in lignin structure between primary and secondary cell wall.

Hybrid aspen (Populus tremula x tremuloides) cell cultures were grown for 7, 14 and 21 days. The cell cultures formed primary cell walls but no secondary cell wall according to carbohydrate analysis and microscopic characterization. The primary walls were lignified, increasingly with age, according to Klason lignin analysis. Presence of lignin in the primary walls, with a higher content in 21-day old cells than in 7-day old cells, was further supported by phloroglucinol/HCl reagent test and confocal microscopy after both immunolocalization and staining with acriflavin. Both laccase and peroxidase activity were found in the cultures and the activity increased during lignin formation. The lignin from the cell culture material was compared to lignin from mature aspen wood, where most of the lignin originates in the secondary cell wall, and which served as our secondary cell wall control. Lignin from the cell walls was isolated and characterized by thioacidolysis followed by gas chromatography and mass spectrometry. The lignin in the cell cultures differed from lignin of mature aspen wood in that it consisted exclusively of guaiacyl units, and had a more condensed structure. Five lignin structures were identified by mass spectrometry in the cell suspension cultures. The results indicate that the hybrid aspen cell culture used in this investigation may be a convenient experimental system for studies of primary cell wall lignin.     

Choline-containing bacteriophage receptors in Streptococcus pneumoniae.

Choline-containing teichoic acid seems to be essential for the adsorption of bacteriophage Dp-1 to pneumococci. This conclusion is based on the following observations: In contrast to pneumococci grown in choline-containing medium, cells grown in medium containing ethanolamine or other submethylated aminoalcohols instead of choline were found to be resistant to infection by Dp-1. Live choline-grown bacteria and heat- or UV-inactivated cells and purified cell walls prepared from these cells were capable of adsorbing phage Dp-1; ethanolamine-grown pneumococci or cell wall preparations were unable to do so. Adsorption of Dp-1 to choline-containing cell walls was competitively inhibited by phosphorylcholine and by several choline-containing soluble cell surface components, such as the Forssman antigen and the teichoic acid-glycan complexes formed by autolytic cell wall degradation. Cell walls prepared from pneumococci grown in ethanolamine or phosphorylethanolamine were inactive. Electron microscopic studies with pneumococci that had segments of choline-containing cell wall material amid ethanolamine-containing regions indicated that the Dp-1 phage particles adsorbed exclusively to the choline-containing surface areas. We suggest that the choline residues of the pneumococcal teichoic acid are essential components of the Dp-1 phage receptors in this bacterium.     

The Chlamydomonas cell wall: characterization of the wall framework

The cell wall of the biflagellate alga Chlamydomonas reinhardtii is a multilayered, extracellular matrix composed of carbohydrates and 20-25 polypeptides. To learn more about the forces responsible for the integrity of this cellulose-deficient cell wall, we have begun studies to identify and characterize the framework of the wall and to determine the effects of the cell wall-degrading enzyme, lysin, on framework structure and protein composition. In these studies we used walls released into the medium by mating gametes. When isolated shed walls are degraded by exogenously added lysin, no changes are detected in the charge or molecular weight of the 20-25 wall proteins and glycoproteins when analyzed on one- and two-dimensional polyacrylamide gels, which suggests that degradation of these shed walls is due either to cleavage of peptide bonds very near the ends of polypeptides or that degradation occurs via a mechanism other than proteolysis. Incubation of walls with Sarkosyl-urea solutions removes most of the proteins and yields thin structures that appear to be the frameworks of the walls. Analysis by polyacrylamide gel electrophoresis shows that the frameworks are highly enriched in a polypeptide of Mr 100,000. Treatment of frameworks with lysin leads to their degradation, which indicates that this part of the wall is a substrate for the enzyme. Although lysin converts the Mr 100,000 polypeptide from an insoluble to a soluble form, there is no detectable change in Mr of the framework protein. Solubilization in the absence of lysin requires treatment with SDS and dithiothreitol at 100 degrees C. These results suggest that the Chlamydomonas cell wall is composed of two separate domains: one containing approximately 20 proteins held together by noncovalent interactions and a second domain, containing only a few proteins, which constitutes the framework of the wall. The result that shed walls can be solubilized by boiling in SDS-dithiothreitol indicates that disulfide linkages are critical for wall integrity. Using an alternative method for isolating walls from mechanically disrupted gametes, we have also shown that a wall-shaped portion of these unshed walls is insoluble under the same conditions in which shed walls are soluble. One interpretation of these results is that wall release during mating and the wall degradation that follows may involve distinct biochemical events.     

Spectrophotometric assay and electrophoretic detection of trans-feruloyl esterase activity.

Wheat bran cell walls were subjected to mild acid hydrolysis and the major phenolic product was purified and identified as 5-O-(trans-feruloyl)-arabinofuranose. Sensitive continuous and stopped, microtiter plate-based spectrophotometric assays for trans-feruloyl esterase activity were developed using this compound as substrate. Procedures were also developed for the detection of trans-feruloyl esterase activities on gels following electrophoresis using this compound. These procedures are applicable to other natural feruloyl esters derived from plant cell walls by enzymatic hydrolysis. The extracellular trans-feruloyl esterases of Aspergillus niger 814 grown on 1% wheat bran were fractionated by anion-exchange chromatography and isoelectric focusing. These studies indicate that there are multiple forms of trans-feruloyl esterase but that most activity is associated with a major isozyme with a pI of 3.2.     

Remodelling of arabinoxylan in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) endosperm cell walls during grain filling

Previous studies using spectroscopic imaging have allowed the spatial distribution of structural components in wheat endosperm cell walls to be determined. FT-IR microspectroscopy showed differing changes in arabinoxylan (AX) structure, during grain development under cool/wet and hot/dry growing conditions, for differing cultivars (Toole et al. in Planta 225:1393–1403, 2007). These studies have been extended using Raman microspectroscopy, providing more details of the impact of environment on the polysaccharide and phenolic components of the cell walls. NMR studies provide complementary information on the types and levels of AX branching both early in development and at maturity. Raman microspectroscopy has allowed the arabinose:xylose (A/X) ratio in the cell wall AX to be determined, and the addition of ferulic acid and related phenolic acids to be followed. The changes in the A/X ratio during grain development were affected by the environmental conditions, with the A/X ratio generally being slightly lower for samples grown under cool/wet conditions than for those from hot/dry conditions. The degree of esterification of the endosperm cell walls with ferulic acid was also affected by the environment, being lower under hot/dry conditions. The results support earlier suggestions that AX is either delivered to the cell wall in a highly substituted form and is remodelled through the action of arabinoxylan arabinofuranohydrolases or arabinofuranosidases, or that low level substituted AX are incorporated into the wall late in cell wall development, reducing the average degree of substitution, and that the rate of this remodelling is influenced by the environment. ¹H NMR provided a unique insight into the chemical structure of intact wheat endosperm cell walls, providing qualitative information on the proportions of mono- and disubstituted AX and the levels of branching of adjacent units. The A/X ratio did not change greatly with either the development stage or the growth conditions, but the ratio of mono- to disubstituted Xylp residues increased markedly (by about fourfold) in the more mature samples, confirming the changes in branching levels determined using FT-IR. To the best of our knowledge, this is the first time that intact endosperm cell walls have been studied by ¹H NMR.     

Cell wall polysaccharides from cell suspension cultures of the Atlantic Forest tree Rudgea jasminoides (Rubiaceae)

Rudgea jasminoides (Rubiaceae) is a tropical tree species native of the Atlantic Forest in the south of Brazil. Previous studies with leaf cell walls of R. jasminoides showed a different proportion of cross-linked glycans compared to what is usually reported for eudicots. However, due to the difficulties of working with whole plant organs, cell suspensions of R. jasminoides, consisting of predominantly undifferentiated cells with mainly primary cell walls, were used to examine cell walls and extracellular soluble polysaccharides (EP) released into the culture medium. Sugar composition and linkage analysis showed homogalacturonans, xylogalacturonans and arabinogalactans to be the predominant EP. In the cell wall, homogalacturonans and arabinogalactans are the major pectins, and xyloglucans and xylans are the major cross-linking glycans. The presence of xylogalacturonans in the R. jasminoides cell cultures seems to be related to the occurrence of a homogeneous cell suspension with loosely attached cells. Although all alkali extractions from the cell walls yielded amounts of xyloglucan that exceed those of the xylans, the latter was found in a proportion that is higher than what has been usually reported for primary cell walls of most eudicots. The xyloglucan from cell walls of cell suspension cultures of R. jasminoides has low fucosylation levels and high proportion of galactosyl residues, a branching pattern commonly found in storage cell-wall xyloglucans.     

Electron Microscopic Observations on the Fine Structure of Cell Walls of Chlamydia psittaci

L-cell cultures were infected with elementary bodies (EB) of meningopneumonitis organisms. Cell walls were prepared from reticulate bodies (RB), which are the intracellular developmental forms into which EB are converted, and from EB at appropriate times after infection. When fragmented EB cell walls were shadowcast with platinum palladium alloy, about one-half of the fragments were seen to be composed of hexagonally arrayed structures on the inner side of the cell wall. When EB cell walls were negatively stained with phosphotungstic acid, they all showed this fine structural array. These macromolecular units were estimated to be about 18 nm in diameter. RB cell walls, harvested at various times after infection, were similarly stained; about 20% of RB walls at 15 hr after infection showed traces of these regular structures, but only 2% of them had the structures at 24 hr. When RB cell walls prepared from penicillin-containing culture were examined, they were observed to be similar to RB without penicillin. When EB cell walls were treated with formamide at 160 C, and then centrifuged in a 10 to 40% potassium tartrate density gradient, hexagonal particles about 20 nm in diameter were obtained as a middle band in the gradient column. These particles were not obtained from RB cell walls harvested from cultures with or without penicillin. It is concluded that the particles are macromolecular subunits located on the inner side of the EB cell walls, that the subunits probably provide the structural rigidity found in the EB, and that their synthesis is inhibited by penicillin.     

Activity of cell-wall degradation associated with differentiation of isolated mesophyll cells of Zinnia elegans into tracheary elements

Cell walls were prepared from cultured mesophyll cells of Zinnia elegans L. that were transdifferentiating into tracheary elements and incubated in a buffer to undergo autolysis. The rate of autolysis of cell walls was determined by measuring the amount of carbohydrate released from the cell walls into the buffer during incubation. During the course of culture of mesophyll cells, the autolysis rate increased markedly at the time when thickenings of secondary cell walls characteristic of tracheary elements became visible (after 48-72 h of culture), and thereafter the rate remained at a high level. Comparative studies on the autolysis rate of cell walls using various control cultures, in which tracheary element differentiation did not take place, revealed a close relationship between the autolysis rate around the 60th hour of culture and differentiation. Sugar analysis by colorimetric assays and gas chromatography of carbohydrates released from the cell walls detected uronic acid, arabinose, galactose, glucose, xylose, rhamnose, fucose, and mannose. Among these sugars, uronic acid was the most abundant, and accounted for approximately half of the total released sugars. The decrease of acidic polysaccharides in the primary cell walls during tracheary element differentiation was visualized by staining cultured cells with alcian blue at pH 2.5. These results suggest that active degradation of components of primary cell walls, including pectin, is integrated into the program of tracheary element differentiation.     

Preparation and Chemical Composition of the Cell Walls of Mature Infectious Dense Forms of Meningopneumonitis Organisms

Relatively large-scale production and purification of meningopneumonitis organisms was developed for chemical and immunological studies on cell walls of the infectious dense forms. By disruption of purified organisms with glass beads in a Mickle shaker, highly purified preparations of cell walls were obtained by sucrose density gradient centrifugation, enzyme digestion, and sodium dodecyl sulfate treatment. The dry-weight recovery of purified cell walls from intact organisms was about 13%. When (32)P-labeled preparations of cell walls were fractionated into acid-soluble, lipid, ribonucleic acid (RNA), deoxyribonucleic (DNA), and residual fractions, about 80% of the (32)P in cell wall preparations was recovered in the phospholipid fraction, which corresponded to about 3% of the total phospholipid in the intact organisms. About 7% of the (32)P in purified cell walls was recovered in the RNA and DNA fractions respectively, but this corresponds to only about 0.4% of the (32)P found in those fractions in intact organisms. From dry-weight determinations, it was calculated that the purified cell wall preparations contained only 0.6% total nucleic acids, and these are probably not true cell wall constituents. These cell walls contained 70 to 75% protein, corresponding to about 14% of the protein in intact organisms. Amino acid analysis of these protein showed the existence of all common amino acids, glucosamine, and galactosamine. However, no muramic acid was detected by the methods employed.     

Pentagalloylglucose down-regulates mast cell surface FcεRI expression in vitro and in vivo

Mast cell activation by immunoglobulin E (IgE)-mediated stimuli is a central event in the pathogenesis of allergic disorders. The present report shows that treatment with pentagalloylglucose (PGG) resulted in a down-regulation of FcεRI surface expression on mucosal-type murine bone marrow-derived mast cells (mBMMCs), which correlated with a reduction in IgE-mediated activation of mBMMCs. Furthermore, PGG prevented development of allergic diarrhea in a food-allergy mouse model and suppressed the up-regulated FcεRI surface expression on mast cells derived from the food-allergy mouse colon. These findings on PGG suggest its therapeutic potential for allergic diseases through suppressing the FcεRI surface expression.     

The ability of land plants to synthesize glucuronoxylans predates the evolution of tracheophytes

Glucuronoxylans with a backbone of 1,4-linked β-D-xylosyl residues are ubiquitous in the secondary walls of gymnosperms and angiosperms. Xylans have been reported to be present in hornwort cell walls, but their structures have not been determined. In contrast, the presence of xylans in the cell walls of mosses and liverworts remains a subject of debate. Here we present data that unequivocally establishes that the cell walls of leafy tissue and axillary hair cells of the moss Physcomitrella patens contain a glucuronoxylan that is structurally similar to glucuronoxylans in the secondary cell walls of vascular plants. Some of the 1,4-linked β-D-xylopyranosyl residues in the backbone of this glucuronoxylan bear an α-D-glucosyluronic acid (GlcpA) sidechain at O-2. In contrast, the lycopodiophyte Selaginella kraussiana synthesizes a glucuronoxylan substituted with 4-O-Me-α-D-GlcpA sidechains, as do many hardwood species. The monilophyte Equisetum hyemale produces a glucuronoxylan with both 4-O-Me-α-D-GlcpA and α-D-GlcpA sidechains, as does Arabidopsis. The seedless plant glucuronoxylans contain no discernible amounts of the reducing-end sequence that is characteristic of gymnosperm and eudicot xylans. Phylogenetic studies showed that the P. patens genome contains genes with high sequence similarity to Arabidopsis CAZy family GT8, GT43 and GT47 glycosyltransferases that are likely involved in xylan synthesis. We conclude that mosses synthesize glucuronoxylan that is structurally similar to the glucuronoxylans present in the secondary cell walls of lycopodiophytes, monilophytes, and many seed-bearing plants, and that several of the glycosyltransferases required for glucuronoxylan synthesis evolved before the evolution of tracheophytes.     

Crown-gall tumors possess tumor-specific antigenic sites on their cell walls

Rabbits were injected with cell walls obtained from crown-gall tumor tissue or the corresponding cell walls from normal potato tissue. The serum obtained from rabbits 53 days after they were injected with tumor cell walls contained immunoglobins that reacted with both tumor and normal cell walls as well as with the cells from the inciting strain of Agrobacterium tumefaciens. When this serum was repeatedly absorbed against normal cell walls and the cells of the inciting strain of Agrobacterium tumefaciens, only tumor-specific immunoglobins remained. These immunoglobins did not react with cell walls obtained from meristematic (nontumorous) potato tissue. Yet this same serum reacted with crown-gall tumor cell walls obtained from turnip and carrot discs.     

A new calcium binding glycoprotein family constitutes a major diatom cell wall component.

Diatoms possess silica-based cell walls with species-specific structures and ornamentations. Silica deposition in diatoms offers a model to study the processes involved in biomineralization. A new wall is produced in a specialized vesicle (silica deposition vesicle, SDV) and secreted. Thus proteins involved in wall biogenesis may remain associated with the mature cell wall. Here it is demonstrated that EDTA treatment removes most of the proteins present in mature cell walls of the marine diatom Cylindrotheca fusiformis. A main fraction consists of four related glycoproteins with a molecular mass of approximately 75 kDa. These glycoproteins were purified to homogeneity. They consist of repeats of Ca2+ binding domains separated by polypeptide stretches containing hydroxyproline. The proteins in the EDTA extract aggregate and precipitate in the presence of Ca2+. Immunological studies detected related proteins in the cell wall of the freshwater diatom Navicula pelliculosa, indicating that these proteins represent a new family of proteins that are involved in the biogenesis of diatom cell walls.     

Effect of Rust Infection on Cell Walls of Barley and Wheat; Immunocytochemistry Using Anti-Barley Thionin as a Probe

Polyclonal antiserum prepared against barley cell wall thionin was used to localize and quantitate immunoreactive material on the cellular level in healthy and rust-infected leaves of barley and wheat. Three types of sites were used for the immunocytochemical analysis: as control sites, mesophyll cell walls were selected in uninoculated leaves, and in leaves that were inoculated with rust but where the sites were not in contact with the pathogen: these were compared with mesophyll cell walls that were in contact with intercellular rust hyphae in inoculated leaves.
Similar amounts of cell wall thionin were detected in all 3 barley cultivars before inoculation. At sites where intercellular hyphae of Puccinia hordei had made contact with mesophyll cell walls, less thionin was found in the compatible host cv. Larker, but in incompatible hosts (cvs. Gold and Bolivia) the thionin concentration did not differ from that of the controls.
Two cultivars of wheat were studied with respect to immunoreactive material in their mesophyll cell walls, the universal rust suscept cv. Little Club and the highly rust-resistant cv. Khapli. Before inoculation, leaves of cv. Khapli contained about twice the amount of immunoreactive material in mesophyll cell walls than those of cv. Little Club. This relation was unchanged in walls that had made contact with P. graminis tritici , but in non-contacted walls of infected cv. Little Club leavest, he concentration of this material had risen to levels typical for those of cv. Khapli. Tests for immunoreactive material with pre-embedding cytochemistry yielded negative results, indicating that it is not exposed on the surface of mesophyll walls in barley and wheat.