Natural lignin modulators improve bagasse saccharification of sugarcane and energy cane in field trials

The burgeoning cellulosic ethanol industry necessitates advancements in enzymatic saccharification, effective pretreatments for lignin removal, and the cultivation of crops more amenable to saccharification. Studies have demonstrated that natural inhibitors of lignin biosynthesis can enhance the saccharification of lignocellulose, even in tissues generated several months post-treatment. In this study, we applied daidzin (a competitive inhibitor of coniferaldehyde dehydrogenase), piperonylic acid (a quasi-irreversible inhibitor of cinnamate 4-hydroxylase), and methylenedioxy cinnamic acid (a competitive inhibitor of 4-coenzyme A ligase) to 60-day-old crops of two conventional Brazilian sugarcane cultivars and two energy cane clones, bred specifically for enhanced biomass production. The resultant biomasses were evaluated for lignin content and enzymatic saccharification efficiency without additional lignin-removal pretreatments. The treatments amplified the production of fermentable sugars in both the sugarcane cultivars and energy cane clones. The most successful results softened the most recalcitrant lignocellulose to the level of the least recalcitrant of the biomasses tested. Interestingly, the softest material became even more susceptible to saccharification.     

EVIDENCE OF DIFFERENCES IN THE DESOXYRIBONUCLEOPROTEIN COMPLEX OF RAPIDLY PROLIFERATING AND NON-DIVIDING CELLS

1. Quantitative cytophotometric analysis of the interphase cells of a rapidly proliferating differentiated tissue such as liver of new born rat, indicates that these cells can be separated into two groups on the basis of their staining characteristics after methanol fixation. 2. These groups are thought to correspond to two stages of interphase. The first, called "autosynthetic interphase," comprises cells which are duplicating chromosomal material in preparation for mitosis, and shows parallel increases in the methyl green and Feulgen staining of DNA and the fast green staining of histone from the diploid (2 C) to double these values (4 C). 3. The second group is designated the "heterosynthetic interphase," during which the cell ceases proliferating and functions in a manner commensurate with its state of differentiation. In this stage Feulgen staining indicates the diploid chromosomal complement, but there is a decreased capacity of the DNA to bind methyl green and of the histone to bind fast green. 4. The difference between the methyl green binding of the heterosynthetic and autosynthetic 2 C cells is due to the presence of a protein in the former which presumably inhibits staining by competing with the dye for binding sites on the DNA. The effect of this inhibition can be removed by extracting the protein, or by blocking the protein basic groups. 5. The decreased fast green staining of histone in the heterosynthetic cells can be minimized by prolonged fixation with formaldehyde. It is thought to stem either from a similar type of inhibition, or from an increased susceptibility of the histone to loss from the cell during this stage. 6. While histone inhibits methyl green staining of DNA in all cells, the differences between the staining properties of the autosynthetic and heterosynthetic interphase cells are believed to be due to another protein, whose properties appear similar to those of the chromosomal "residual protein." It is concluded that a complex of DNA and residual protein existing during the heterosynthetic interphase is dissociated during the autosynthetic interphase.     

Escherichia coli Heat Shock Protein DnaK: Production and Consequences in Terms of Monitoring Cooking

Through use of commercially available DnaK proteins and anti-DnaK monoclonal antibodies, a competitive enzyme-linked immunosorbent assay was developed to quantify this heat shock protein in Escherichia coli ATCC 25922 subjected to various heating regimens. For a given process lethality (F₇₀¹⁰ of 1, 3, and 5 min), the intracellular concentration of DnaK in E. coli varied with the heating temperature (50 or 55°C). In fact, the highest DnaK concentrations were found after treatments at the lower temperature (50°C) applied for a longer time. Residual DnaK after heating was found to be necessary for cell recovery, and additional DnaK was produced during the recovery process. Overall, higher intracellular concentrations of DnaK tended to enhance cell resistance to a subsequent lethal stress. Indeed, E. coli cells that had undergone a sublethal heat shock (105 min at 55°C, F₇₀¹⁰ = 3 min) accompanied by a 12-h recovery (containing 76,786 ± 25,230 molecules/cell) resisted better than exponentially growing cells (38,500 ± 6,056 molecules/cell) when later heated to 60°C for 50 min (F₇₀¹⁰ = 5 min). Results reported here suggest that using stress protein to determine cell adaptation and survival, rather than cell counts alone, may lead to more efficient heat treatment.     

Intramolecular electron transfer in the oxidation of amines by methylamine oxidase from Arthrobacter P1

 The intramolecular electron-transfer rate constant for the Cu(II)–topa_NH2⇌ Cu(I)–topa_SQ equilibrium in methylamine oxidase has been measured by temperature-jump relaxation techniques. At pH 7.0 the estimated k_obs = 150±30 s^–1 for both methylamine and benzylamine; assuming the equilibrium constant is ≈0.7–1 at pH 7.0 and 296 K, this would correspond to a forward electron-transfer rate constant k_ET≈ 60–75 s^–1. Although substantially slower than the previously determined k_ET≈ 20 000 s^–1 for pea seedling amine oxidase [5] steady-state kinetics measurements established that k_ET > k_cat≈ 4–10 s^–1. Thus the Cu(I)-semiquinone state is a viable intermediate in methylamine oxidase turnover. Received: 16 August 1995 / Accepted: 21 December 1995     

Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non-redundant ESTs

Nearly 7000 Arabidopsis thaliana -expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non-redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.     

Poplar (Populus nigra L.) plants transformed with aBacillus thuringiensis toxin gene: insecticidal activity and genomic analysis

Insect-resistant poplar (Populus nigra L.) plants have been produced by infecting leaves withAgrobacterium tumefaciens strains carrying a binary vector containing different truncated forms of aBacillus thuringiensis (B.t.) toxin gene under a duplicated CaMV 35S promoter. Putative transgenic plants were propagated by cuttings at two experimental farms (in Beijing and Xinjiang, China). At 2–3 years after transformation, 17 of them were selected on the bases of insect-tolerance and good silvicultural traits, and evaluated for insect resistance, for the presence of theB.t. toxin DNA fragment (Southern blots and PCR) and for the expression of the transgene (western and northern blots). Somaclonal variation, as suggested by the appearance of permanent changes in the shape of the leaves, was also investigated with molecular tools (RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA) and microsatellite DNA).Bioassays withApochemia cineraius andLymantria dispar on the leaves of the selected clones showed different and, in some cases, high levels of insecticidal activity. The molecular analysis demonstrated integration and expression of the foreign gene. Somatic changes were correlated to extensive genomic changes and were quantified in dendrograms, in terms of genomic similarity. The analysis of control plants suggested that genomic changes were correlated to thein vitro culture step necessary forA. tumefaciens-mediated gene transfer, rather than to the integration of the foreign genes.Three transgenic clones (12, 153 and 192), selected for insect resistance, reduced morphological changes and promising silvicultural traits, are now under large-scale field evaluation in six different provinces in China.     

Does photorespiration protect the photosynthetic apparatus in french bean leaves from photoinhibition during drought stress?

Dithiothreitol (DTT), an inhibitor of violaxanthin de-epoxidation and zeaxanthin formation in chloroplasts, inhibited blue-light-stimulated stomatal opening in epidermal peels of Vicia faba L. in a concentration-dependent fashion. Complete inhibition was observed at 3 mM DTT. The DTT effect was specific for the stomatal response to blue light, and the red-light-stimulated opening, which depends on photosynthetic reactions in the guard cells, was unaffected. Preirradiation of stomata in epidermal peels with increasing photon fluence rates of red light, prior to an incubation in 10 mol·m^-2·s^-1 of blue light and 100 mol·m^-2·s^-1 red light, resulted in a DTT-sensitive, blue-light-stimulated opening that was proportional to the fluence rate of the red light pre-treatment. Guard cells in epidermal peels and guard-cell protoplasts irradiated with red light showed increases in their zeaxanthin content that depended on the fluence rate of red light, or on the incubation time. The increases in zeaxanthin concentration were inhibited by DTT. The obtained results indicate that zeaxanthin could function as a photoreceptor mediating the stomatal responses to blue light.Abbreviation DTT dithiothreitol This work was supported by grants from the National Science Foundation and the US Department of Energy to E.Z.     

Laminin and Neuropeptide Y Are Increased by Synapsin Transfection in Cultured NG108-15 Neuroblastoma/Glioma Hybrid Cells

Abstract: We have investigated the presence and expression of laminin and neuropeptide Y (NPY) in several NG108-15 cell lines transfected with synapsin Ib, IIa, or IIb. The content of laminin, a basal membrane glycoprotein that promotes adhesion and induces neurite outgrowth and neuronal differentiation, was increased in all transfected cell lines examined. In cells that were chemically differentiated with prostaglandin E₁ plus 3-isobutyl-1-methylxanthine, laminin levels were increased even further. The content of NPY, suggested to be a neurotransmitter/neuromodulator in peripheral sympathetic neurons as well as in central neurons, was also increased in all transfected cell lines examined. Immunohistochemical analysis combined with confocal laser microscopy showed that NPY staining was granular and very often enriched in neuritic varicosities. The distribution and the staining pattern of NPY were consistent with storage of NPY in large dense-cored vesicles. The results indicate that, in differentiated neurons, the synapsins increase the levels of a neuropeptide transmitter stored in large dense-cored vesicles and of an extracellular matrix protein associated with neuronal maturation.     

Swim Stress Increases the Potency of Glycine at the N-Methyl-d-Aspartate Receptor Complex

Abstract: We have previously demonstrated that chronic administration of antidepressants results in a reduction in the potency of glycine to displace 5,7-[³H]dichlorokynurenic acid (5,7-[³H]-DCKA) from the strychnine-insensitive glycine recognition site of the NMDA receptor complex. We now report that exposure of rats to the forced swim test results in a 56% increase in the potency of glycine to displace 5,7-[³H]DCKA from frontal cortical homogenates. These data are consistent with the hypothesis that the forced swim test, a preclinical screen sensitive to acute administration of antidepressant drugs and NMDA receptor antagonists, also results in adaptation of the NMDA receptor complex. Moreover, these data lend further support to the hypothesis that glutamatergic pathways are involved in the neurobiological response to stress and, potentially, in the pathophysiology of depression.     

Intramolecular domain-domain interactions and intermolecular self-association in bovine prothrombin. A potentiometric and laser light-scattering study

The interaction of bovine prothrombin with Ca²⁺ and Mg²⁺ ions was investigated by following H⁺ release as a function of metal ion concentration at pH 6 and pH 7.4 at high and low ionic strength. Prothrombin Ca²⁺ and Mg²⁺ binding is characterized by high- and low-affinity sites. M²⁺ binding at these sites is associated with intramolecular conformational changes and also with intermolecular self-association. The pH dependence of H⁺ release by M²⁺ is bell shaped and consistent with controlling pK_a values of 4.8 and 6.5. At pH 6 and low ionic strength, both Ca²⁺ and Mg²⁺ titrations following H⁺ release clearly show independent low- and high-affinity binding sites. Laser light scattering reveals that at pH 7.4 and low ionic strength, and at pH 6.0 and high ionic strength, the prothrombin molecular weight is between 73 and 98 kD. At pH 7.4 and high ionic strength, prothrombin is monomeric in the absence of metal ions, but appears to dimerize in the presence of M²⁺. At pH 6.0 and low ionic strength prothrombin exists as a dimer in the absence of metal ions and is tetrameric in the presence of Ca²⁺ and remains dimeric in the presence of Mg²⁺. These results and those for metal ion-dependent H⁺ release indicate that H⁺ release occurs concomitantly with association processes involving prothrombin.Abbreviations GLA -carboxyglutamic acid; fragment 1. amino terminal residues 1–156 of bovine prothrombin - MES 2-(N-morpholino) ethanesulfonic acid - MOPS 3-(N-morpholino) propanesulfonic acid - PS/PC phosphatidylserine/phosphatidylcholine vesicles - ionic strength