Stereoisomeric guaiacylglycerol-β-coniferyl aldehyde ether induces distinctive apoptosis by downregulation of MEK/ERK pathway in hepatocellular carcinoma cells

Two 8-O-4′-type neolignan epimers erythro-guaiacylglycerol-β-coniferyl aldehyde ether (1) and threo-guaiacylglycerol-β-coniferyl aldehyde ether (2) were isolated from the stems of Picrasma quassioides. Further chiral separation gave two pairs of enantiomers 1a/1b and 2a/2b. The cytotoxicity assay against hepatocellular carcinoma Hep3B and HepG2 cells was evaluated by MTT assay. The results showed that 1b (IC₅₀ = 45.56 μM) and 2b (IC₅₀ = 39.02 μM) had more cytotoxic effect than its enantiomers 1a (IC₅₀ = 82.66 μM) and 2a (IC₅₀ = 67.97 μM) in Hep3B cells, respectively. Moreover, 1b and 2b could induce more apoptotic cells as well as higher reactive oxygen species (ROS) generation than 1a and 2a at 50 μM. In addition, a further study on the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways was investigated. The results revealed that all compounds had no significant effect on PI3K/AKT pathway, however, 1b and 2b attenuated the relative levels of p-MEK and p-ERK when compared with 1a and 2a. Taken together, the absolute configurations of guaiacylglycerol-β-coniferyl aldehyde ether had an impact on the inhibitory effect on Hep3B cells. The inactivation of MEK/ERK signaling pathway might contribute to apoptosis induction and ROS generation in 1b- and 2b-treated cells.     

Metabolism of the lignin model compounds veratrylglycerol-β-guaiacyl ether and 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether by Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium metabolized the lignin model compounds veratylglycerol--guaiacyl ether I and 4-ethoxy-3-methoxy-phenylglycerol--guaiacyl ether V in stationary culture under an atmosphere of 100% oxygen and under nitrogen limiting conditions. 2-(o-methoxyphenoxy)-ethanol VII was identified as a product of the metabolism of both substrates. Veratryl alcohol and 4-ethoxy-3-methoxybenzyl alcohol IV were identified as metabolites of I and V respectively. Metabolites were identified after comparison with chemically synthesized standards by mass spectrometry. These results indicate the existence of an enzyme system capable of directly cleaving the etherated dimers I and V at the , bond. The additional identification of 2-(o-methoxyphenoxy)-1,3 propanediol IX as a metabolic product indicates that cleavage of the alkyl-phenyl bond of these dimers or their metabolites also occurs.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC Thin layer chromatography     

Alkyl-phenyl cleavage of the lignin model compounds guaiacylglycol and glycerol-β-guaiacyl ether by Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized guaiacylglycol--guaiacyl ether (I) in high nitrogen, shaking and stationary cultures. 2-(o-Methoxyphenoxy) ethanol (X), 2-(o-methoxyphenoxy) acetic acid (IX) and methoxy-phydroquinone (MHQ) were identified as products of the metabolism of (I). P. chrysosporium also metabolized guaiacylglycerol--guaiacyl ether (IV) in high nitrogen stationary cultures. 2-(o-Methoxyphenoxy)-1,3 propanediol (XII) and 3-hydroxy, 2-(o-methoxy-phenyxy) propionic acid (XIV) were identified as products of the metabolism of (IV). Finally, P. chrysosporium metabolized -deoxyguaiacylglycol--guaiacyl ether (VI) and -deoxyguaiacylglycerol--guaiacyl ether (VII) in limiting nitrogen cultures. 2-(o-Methoxyphenoxy) ethanol (X) and 2-(o-methoxyphenoxy)-1,3 propanediol (XII) were identified as products of the metabolism of VI and VII respectively indicating hydroxylation of those substrates with subsequent alkyl-phenyl bond cleavage. Metabolites were identified after comparison with chemically synthesized standards by GLC-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography - MHQ methoxyhydroquinone     

Degradation of arylglycerol-β-aryl ethers,lignin substructure models,by Fusarium solani

Degradation of arylglycerol--aryl ethers, the most important substructure in lignin, by Fusarium solani M-13-1 was investigated. The fungus was shake-cultured in mineral salts media which contained either guaiacylglycerol--vanillic acid ether (2), syringylglycerol--vanillin ether (4), veratrylglycerol-vanillin ether (17) or glycerol-2-vanillic acid ether (9) as sole carbon source. Culture filtrates from incubations with 4 contained syringylglycerol-vanillic acid ether (6), 9 and 2,6-dimethoxy-p-benzoquinone (16). Culture filtrates from incubations with 2 also contained 9. Veratrylglycerol--vanillic acid ether (18) derived from 17 was not metabolized further. These results inidicate that the alkyl-aryl C-C bond in both 2 and 5 was cleaved by phenol oxidizing enzymes with formation of 9 and methoxy-p-benzoquinone (15 and 16). Compound 9 was converted to glycerol-2-vanillic acid ether monoacetate (10), glyceric acid-2-vanillic acid ether (11) and ethylene glycol monovanillic acid ether (12).Non-Standard Abbreviations Ar aromatic - THF tetrahydrofuran - TLC thin layer chromatography     

β-Ether cleavage of the lignin model compound 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether and derivatives by Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized 4-ethoxy-3-methoxyphenyl-glycerol--guaiacyl ether (V) in low nitrogen, stationary cultures under which conditions the ligninolytic enzyme system is expressed. 4-Ethoxy-3-methoxyphenylglycerol XIII, guaicol and 4-ethoxy-3-methoxybenzyl alcohol (II) were isolated as metabolic products. Exogenously added XIII was rapidly converted to 4-ethoxy-3-methoxybenzyl alcohol indicating that it is an intermediate in the metabolism of V. P. chrysosporium also metabolized 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-3-hydroxypropane VI. The degradation pathway for this dimer also included initial -ether cleavage and -hydroxylation of the diol product 1-(4-ethoxy-3-methoxyphenyl) 2,3 dihydroxypropane (XI) to yield the triol XIII which was cleaved at the , bond to yield 4-ethoxy-3-methoxybenzyl alcohol. Finally P. chrysosporium also cleaved the dimer 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1-hydroxypropane (VIII) at the -ether linkage yielding 1-(4-ethoxy-3-methoxyphenyl) 1,2 dihydroxypropane (IX) which was subsequently cleaved at the , bond to yield II. All of the results indicate that oxidative -ether cleavage is an important initial reaction in the metabolism of -aryl ether lignin substructure dimeric compounds. Metabolities were identified after comparison with chemically synthesized standards by gas liquid chromatography-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography     

Lytic polysaccharide monooxygenases promote oxidative cleavage of lignin and lignin–carbohydrate complexes during fungal degradation of lignocellulose

Overcoming lignocellulosic biomass recalcitrance, especially the cleavage of cross-linkages in lignin–carbohydrate complexes (LCCs) and lignin, is essential for both the carbon cycle and industrial biorefinery. Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in fungal polysaccharide oxidative degradation. Nevertheless, comprehensive analysis showed that LPMOs from a white-rot fungus, Pleurotus ostreatus, correlated well with the Fenton reaction and were involved in the degradation of recalcitrant nonpolysaccharide fractions in this research. Thus, LPMOs participated in the extracellular Fenton reaction by enhancing iron reduction in quinone redox cycling. A Fenton reaction system consisting of LPMOs, hydroquinone, and ferric iron can efficiently produce hydroxy radicals and then cleave LCCs or lignin linkages. This finding indicates that LPMOs are underestimated auxiliary enzymes in eliminating biomass recalcitrance.     

p-Benzoquinone monoketals,novel degradation products of β-O-4 lignin model compounds by Coriolus versicolor and lignin peroxidase of Phanerochaete chrysosporium

2-(4-Ethoxy-3-methoxyphenyl)-3-hydroxymethyl-6,10-dimethoxy-1,4-dioxaspiro[4,5]deca-6,9-diene-8-one (III) and its isomer IV were identified as catabolites of 4-ethoxy-3-methoxyphenylglycerol-β-syringaldehyde ether (I) by the culture of Coriolus versicolor. Compound III was also produced from 4-ethoxy-3-methoxyphenylglycerol-β-syringic acid ether (II) by lignin peroxidase of Phanerochaete chrysosporium. An isotopic experiment showed that molecular oxygen was incorporated into the quinone oxygen of III in the degradation of II by lignin peroxidase.     

Aggregation and collapse of fungal wall vesicles in hyphal tips: a model for the origin of the Spitzenk?rper

The intracellular origins of polarity and branch initiation in fungi centre upon a localization in the supply of fungal wall constituents to specific regions on the hyphal wall. Polarity is achieved and maintained by accumulating secretory vesicles, prior to incorporation into the wall, in the form of an apical body or Spitzenkörper. However, neither the mechanisms leading to this accumulation nor the initiation of branching, are as yet understood. We propose a mechanism, based on experimental evidence, which considers the mechanical properties of the cytoskeleton in order to explain these phenomena. Cytoskeletal viscoelastic forces are hypothesized to be responsible for biasing vesicles in their motion, and a mathematical model is derived to take these considerations into account. We find that, as a natural consequence of the assumed interactions between vesicles and cytoskeleton, wall vesicles aggregate in a localized region close to the tip apex. These results are used to interpret the origin of the Spitzenkörper. The model also shows that an aggregation peak can collapse and give rise to two new centres of aggregation coexisting near the tip. We interpret this as a mechanism for apical branching, in agreement with published observations. We also investigate the consequences and presumptive role of vesicle–cytoskeleton interactions in the migration of satellite Spitzenkörper. The results of this work strongly suggest that the formation of the Spitzenkörper and the series of dynamical events leading to hyphal branching arise as a consequence of the bias in vesicle motion resulting from interactions with the cytoskeleton.     

Degradation of d,l-syringaresinol,a β-β′ linked lignin model compound,by Fusarium solani M-13-1

A - linked lignin model compound, d,l-syringaresinol monobenzyl ether (Ib) was incubated with Fusarium solani M-13-1 in a shaking culture. From the culture filtrates, three compounds II, IIIb and IV were isolated and identified. Substrate Ib was oxidized at the -position of the side chain to give a hemiketal, an -hydroxylated compound IIA, which was then transformed to the ketoalcohol, 3-hydroxymethyl-2-(4-benzyloxy-3,5-dimethoxyphenyl)-4-(4-hydroxy-3,5-dimethoxybenzoyl)-tetrahydrofuran (IIB). These products were converted to a -lactone derivative, 6-oxo-2-(4-benzyloxy-3,5-dimethoxyphenyl)-3,7-dioxabicyclo-[3,3,0]-octane (IIIb), via alkyl-aryl cleavage. The syringyl moiety released from II by the cleavage reaction was identified as 2,6-dimethoxy-p-benzoquinone (IV). Incubation of 2,6-dimethoxyphenol (V) in fungal culures did not give the p-quinone IV. d,l-Syringaresinol dimethyl ether was not degraded and the etherated moiety of Ib was not attacked by the fungus, indicating that the degradation of d,l-syringaresinol was catalyzed by phenol oxidizing enzymes. The oxidation products of Ib with peroxidase/H₂O₂ was investigated and discussed in relation to the degradation products of the fungus.Abbreviation TLC thin layer chromatography     

Occurrence of a Putative Precursor to the Amyloplast,the “Amyloplast Initial” in Developing Stolons of the Potato

Cells at the apical part of developing stolons of the potato (Solatium tuberosum L. cv. Norin 1) were analyzed for the occurrence of putative precursors to amyloplasts, designated “amyloplast initial.” Ultrastructural studies showed that the cells contained the expected novel organelle. It was about 1 μm in diameter, devoid of thylakoid membranes, and was stained to a similar extent as the stroma of amyloplasts by uranyl acetate and lead citrate. Formation of thylakoid membranes and starch granules takes place at an early stage of development of these initials when they are just a few μm in diameter. At this stage, proliferation of the initials takes places by division at random sites.     

Secretion of a heat-stable fungalβ-glucanase from transgenic,suspension-cultured barley cells

Transgenic plant cell cultures have a potential for production and secretion of important proteins and peptides. To assess the possibilities of using a stable barley suspension culture for secretion of heterologous proteins in active form, we expressed the cDNA of the thermostable-glucanase (EGI) ofTrichoderma reesei in barley suspension cells. The cDNA coding for EGI and its signal sequence was placed under the control of the CaMV 35S promoter and the construction was transferred to the cells by particle bombardment. Stably transformed lines were obtained by selecting for a cotransformed antibiotic resistance marker. The expression of EGI cDNA led to accumulation of EGI in the culture medium, as shown by analysis with EGI-specific antibodies. Enzymatic assays confirmed that the EGI secreted by the suspension cells retained its activity and thermostable character. Furthermore, it was shown that the enzyme produced by the transgenic suspension culture could be used for degradation of soluble-glucans during mashing.     

Anticooperativity in diffusion-controlled reactions with pairs of anisotropic domains: a model for the antigen–antibody encounter

The encounter between anisotropic agents in diffusion-controlled reactions is a topic of very general relevance in chemistry and biology. Here we introduce a simplified model of encounter of an isotropic molecule with a pair of partially reacting agents and apply it to the encounter reaction between an antibody and its antigen. We reduce the problem to the solution of dual series relations, which can be solved iteratively, yielding the exact solution for the encounter rate constant at any desired order of accuracy. We quantify the encounter effectiveness by means of a simple indicator and show that the two binding centers systematically behave in an anti-cooperative fashion. However, we demonstrate that a reduction of the binding active sites allows the composite molecule to recover binding effectiveness, in spite of the overall reduction of the rate constant. In addition, we provide a simple formula that enables one to calculate the anti-cooperativity as a function of the size of the binding site for any values of the separation between the two active lobes and of the antigen size. Finally, some biological implications of our results are discussed.     

Synthesis and degradation of poly-β-hydroxybutyrate in a sequencing batch biofilm reactor

The aim of this work was the study of poly-β-hydroxybutyrate (PHB) formation and degradation in a sequencing batch biofilm reactor (SBBR). The SBBR was operated in cycles comprising three individual phases: mixed fill, aeration and draw. A synthetic substrate solution with acetate and ammonium was used.PHB was formed during the aeration phase immediately after acetate depletion, and was subsequently consumed for biomass growth, owing to the high oxygen concentration in the reactor. It was observed a combination of suspended and biofilm growth in the SBBR with predominance of the fixed form of biomass (506 Cmmol and 2102 Cmmol, respectively). Maximum PHB fraction of suspended biomass (0.13 Cmol/Cmol) was considerably higher than that of biofilm (0.01 Cmol/Cmol). This may possibly be explained by a combination of two factors: lower mass transfer limitation of acetate and higher fraction of heterotrophs in suspended biomass compared to the ones of biofilm.     

Ultrastructure of the endocrine cell types in the adenohypophysis of the teleost,Poecilia latipinna — a morphometric model

Summary A morphometric model providing detailed quantitative information on the ultrastructure of the adenohypophysial endocrine cells has been developed for Poecilia latipinna. The model consists of various morphological components quantified in terms of volumes, surfaces or numbers. For prolactin and growth hormone cells, appropriate results are expressed relative to the average volume of that cell type. The difficulties of quantifying EM data on pituitary glands, together with the various sources of error to which the data are clearly open, have been discussed. Some practical applications of quantitative EM to problems in fish pituitary research are outlined.I thank Dr. J.N. Ball for supplying the fish and Mr. L. Ethridge for technical assistance.     

Does the Initial Floristic Composition model of succession really work?

Harvey & Holzman, in this issue of the Journal of Vegetation Science, describe divergent successional pathways after fire in Pinus muricata forest. Fourteen years after fire, the shrub Ceanothus thyrsiflorus had occupied flat parts of the area. They predicted it would continue to prevent succession to P. muricata forest, as in the initial floristic composition concepts of Clements and Egler.     

Expression of a library of fungal β-glucosidases in Saccharomyces cerevisiae for the development of a biomass fermenting strain

Converting cellulosic biomass to ethanol involves the enzymatic hydrolysis of cellulose and the fermentation of the resulting glucose. The yeast Saccharomyces cerevisiae is naturally ethanologenic, but lacks the enzymes necessary to degrade cellulose to glucose. Towards the goal of engineering S. cerevisiae for hydrolysis of and ethanol production from cellulose, 35 fungal β-glucosidases (BGL) from the BGL1 and BGL5 families were screened for their ability to be functionally expressed and displayed on the cell surface. Activity assays revealed that the BGL families had different substrate specificities, with only the BGL1s displaying activity on their natural substrate, cellobiose. However, growth on cellobiose showed no correlation between the specific growth rates, the final cell titer, and the level of BGL1 activity that was expressed. One of the BGLs that expressed the highest levels of cellobiase activity, Aspergillus niger BGL1 (Anig-Bgl101), was then used for further studies directed at developing an efficient cellobiose-fermenting strain. Expressing Anig-Bgl101 from a plasmid yielded higher ethanol levels when secreted into the medium rather than anchored to the cell surface. In contrast, ethanol yields from anchored and secreted Anig-Bgl101 were comparable when integrated on the chromosome. Flow cytometry analysis revealed that chromosomal integration of Anig-Bgl101 resulted in a higher percentage of the cell population that displayed the enzyme but with overall lower expression levels.     

Can co-culturing of two white-rot fungi increase lignin degradation and the production of lignin-degrading enzymes?

The aim of this work was to investigate the poorly understood effects of co-culturing of two white rot fungi on the production of lignin-degrading enzyme activities. Four species, Ceriporiopsis subvermispora, Physisporinus rivulosus, Phanerochaete chrysosporium and Pleurotus ostreatus were cultured in pairs to study the degradation of aspen wood and the production of lignin-degrading enzymes. Potential of co-culturing for biopulping was evaluated. Chemical analysis of decayed aspen wood blocks showed that co-culturing of C. subvermispora with P. ostreatus could significantly stimulate wood decay, when compared to monocultures. Based on the fungi tested here, however, this effect is species-specific. Other combinations of fungi were slightly stimulating or not stimulatory. The pattern of lignin degradation was altered towards the acid insoluble part of lignin especially in co-cultures where P. ostreatus was included as a partner. The use of agar plates containing the polymeric dye Poly R-478 showed elevated dye decolourization at the confrontation zone between mycelia. Laccase was significantly stimulated only in the co-culture of P. ostreatus with C. subvermispora. Manganese peroxidase activity was stimulated in co-cultures of P. ostreatus with C. subvermispora or with P. rivulosus. Immunoblotting indicated changes in lignin-degrading enzymes and/or their isoform composition in response to co-culturing. This is the first report on the effects of co-culturing of potential biopulping fungi on wood degradation, and gives basic knowledge on fungal interactions during wood decay that can be utilized in practical applications.     

On the role of the monolignol γ-carbon functionality in lignin biopolymerization

In order to investigate the importance of the monomeric γ-carbon chemistry in lignin biopolymerization and structure, synthetic lignins (dehydrogenation polymers; DHP) were made from monomers with different degrees of oxidation at the γ-carbon, i.e., carboxylic acid, aldehyde and alcohol. All monomers formed a polymeric material through enzymatic oxidation. The polymers displayed similar sizes by size exclusion chromatography analyses, but also exhibited some physical and chemical differences. The DHP made of coniferaldehyde had poorer solubility properties than the other DHPs, and through contact angle of water measurement on spin-coated surfaces of the polymeric materials, the DHPs made of coniferaldehyde and carboxylic ferulic acid exhibited higher hydrophobicity than the coniferyl alcohol DHP. A structural characterization with ¹³C NMR revealed major differences between the coniferyl alcohol-based polymer and the coniferaldehyde/ferulic acid polymers, such as the predominance of aliphatic double bonds and the lack of certain benzylic structures in the latter cases. The biological role of the reduction at the γ-carbon during monolignol biosynthesis with regard to lignin polymerization is discussed.     

Computational analysis reveals the coupling between bistability and the sign of a feedback loop in a TGF-β1 activation model

Background

Bistable behaviors are prevalent in cell signaling and can be modeled by ordinary differential equations (ODEs) with kinetic parameters. A bistable switch has recently been found to regulate the activation of transforming growth factor-β1 (TGF-β1) in the context of liver fibrosis, and an ordinary differential equation (ODE) model was published showing that the net activation of TGF-β1 depends on the balance between two antagonistic sub-pathways.

Results

Through modeling the effects of perturbations that affect both sub-pathways, we revealed that bistability is coupled with the signs of feedback loops in the model. We extended the model to include calcium and Krüppel-like factor 2 (KLF2), both regulators of Thrombospondin-1 (TSP1) and Plasmin (PLS). Increased levels of extracellular calcium, which alters the TSP1-PLS balance, would cause high levels of TGF-β1, resembling a fibrotic state. KLF2, which suppresses production of TSP1 and plasminogen activator inhibitor-1 (PAI1), would eradicate bistability and preclude the fibrotic steady-state. Finally, the loop PLS???TGF-β1???PAI1 had previously been reported as negative feedback, but the model suggested a stronger indirect effect of PLS down-regulating PAI1 to produce positive (double-negative) feedback in a fibrotic state. Further simulations showed that activation of KLF2 was able to restore negative feedback in the PLS???TGF-β1???PAI1 loop.

Conclusions

Using the TGF-β1 activation model as a case study, we showed that external factors such as calcium or KLF2 can induce or eradicate bistability, accompanied by a switch in the sign of a feedback loop (PLS???TGF-β1???PAI1) in the model. The coupling between bistability and positive/negative feedback suggests an alternative way of characterizing a dynamical system and its biological implications.

     

Study of a specific lignin model: γ-oxidation and how it influences the hydrolysis efficiency of alcohol-aldehyde dehydrogenation copolymers

Six coniferyl alcohol-coniferaldehyde dehydrogenation copolymers (DHcoPs) were synthesized in order to determine the influence of an increased number of aldehyde functions on hydrolysis. After heterogeneous hydrolysis using acidic Montmorillonite K10 clay, the DHcoPs were thioacidolyzed and analyzed by gel permeation chromatography (GPC). Comparison of the thioacidolyzed products, with and without the hydrolysis step, showed that there was a greater proportion of condensation reaction in the absence of aldehyde. When the coniferaldehyde content in the initial synthetic mixture was more than 30% (w/w), only a low fraction of condensed products was generated during the K10 clay hydrolysis step. This suggests that condensation pathways are mainly due to the alcohol present in the γ-position in the DHcoPs. Investigation of the reactivity and the potential condensation of aldehyde and alcohol monomers under hydrolysis conditions showed the important conversion of coniferyl alcohol and conversely the stability of coniferaldehyde.