Low temperature alkali pretreatment for improving enzymatic digestibility of sweet sorghum bagasse for ethanol production

A low temperature alkali pretreatment method was proposed for improving the enzymatic hydrolysis efficiency of lignocellulosic biomass for ethanol production. The effects of the pretreatment on the composition, structure and enzymatic digestibility of sweet sorghum bagasse were investigated. The mechanisms involved in the digestibility improvement were discussed with regard to the major factors contributing to the biomass recalcitrance. The pretreatment caused slight glucan loss but significantly reduced the lignin and xylan contents of the bagasse. Changes in cellulose crystal structure occurred under certain treatment conditions. The pretreated bagasse exhibited greatly improved enzymatic digestibility, with 24-h glucan saccharification yield reaching as high as 98% using commercially available cellulase and β-glucosidase. The digestibility improvement was largely attributed to the disruption of the lignin-carbohydrate matrix. The bagasse from a brown midrib (BMR) mutant was more susceptible to the pretreatment than a non-BMR variety tested, and consequently gave higher efficiency of enzymatic hydrolysis.     

Contrasting of Lowicryl K4M thin sections

Summary A method is presented for increasing the contrast of cellular structures on ultrathin sections from tissues embedded in Lowicryl K4M. The method, designated UA/MC adsorption staining, is based on the uranyl acetate/methyl cellulose staining of thawed cryosections. Ultrathin Lowicryl K4M sections were exposed to a uranyl acetate/methyl cellulose solution and the excess solution was removed with filter paper, leaving the remainder to air dry on the section. Sections on the grids were then directly observed in the electron microscope. Parameters such as methyl cellulose and uranyl acetate concentrations, duration of staining, temperature and pH were all assessed for their effect on subsequent contrast formation. Conditions were achieved which yielded intense contrast of cellular membranes, basement membranes and extracellular matrix components usually not apparent in Lowicryl K4M thin sections routinely counter-stained with uranyl acetate and lead acetate. The enhancement of the contrast of these structures does not obscure colloidal gold particles used for immunocytochemistry or lectin labeling, thus making the UA/MC adsorption staining method useful for increasing membrane contrast in routine post-embedding immuno- and lectin cytochemistry on Lowicryl K4M thin sections.     

Studies on Proteolytic Enzyme of the Cricket,Gryllulus taiwanemma Ohmachi

A protease was isolated from the alimentary canal of crickets. This cricket protease was purified by ammonium sulfate, rivanol and acetone fractionations, and DEAE-cellulose and CM-cellulose (Ca-form) column chromatographies. The optimum temperature was 50°C and the optimum pH was 8.0. For preservation, the enzyme was most stable at pH 3.0. Aluminum had the best stabilizing action with no drops of enzyme activity after 24 hours of dialysis. The cricket protease was specific for the synthetic substrate, α-benzoyl-l-arginine amide. Cricket protease had a K_m. 10³ of 2.8 which is similar to that of trypsin, 1.2. The Ea was 3,770 while that of trypsin was 14,960 using α-benzoyl-l-arginine amide as the substrate. Although cricket protease has the same substrate specificity and similar optimum pH and pH-stability as trypsin, it differed in metal requirements to obtain activity. Certain metals are essential for cricket protease activity.     

Genome position and gene amplification

Background  

Amplifications, regions of focal high-level copy number change, lead to overexpression of oncogenes or drug resistance genes in tumors. Their presence is often associated with poor prognosis; however, the use of amplification as a mechanism for overexpression of a particular gene in tumors varies. To investigate the influence of genome position on propensity to amplify, we integrated a mutant form of the gene encoding dihydrofolate reductase into different positions in the human genome, challenged cells with methotrexate and then studied the genomic alterations arising in drug resistant cells.     

Fine Structure of Bacillus subtilis : II. Sporulation Progress

The sporulation process in Bacillus subtilis has been studied principally with KMnO₄ fixation, but also, for the purpose of comparison, with OsO₄ and mixtures of both fixatives. At a very early stage, the pre-spore is seen to consist of what seems to be the nuclear material and granular substance, surrounded by a layer of dense material destined to become the innermost layer of the spore coat. At a subsequent stage, a light interspace is observed that is destined to become the spore cortex. The mature spore shows a very complex structure. The spore coat is composed of three layers, the middle layer of which consisted of 5 to 8 lamellae of thin membranes and interspaces, both about 20 to 25 A thick. Between the inner layer of the spore coat and the spore cortex, a thin membrane with an affinity to the cortex can be observed. The spore coat is enclosed within two envelopes, one loosely surrounding the core, and the other adhering to it. The process of spore maturation has been studied in detail. Certain peculiar cellular structures have been observed that seemed to represent features of abnormal sporulation processes.     

Pretreatment of microcrystalline cellulose flakes with CaCl2 increases the surface area, and thus improves enzymatic saccharification

Glucose production from cellulose flakes with cellulases was improved after pretreatment with saturated CaCl2 at room temperature. When pretreated microcrystalline cellulose flakes (Funacel II, Funakoshi Co., Ltd, Tokyo, Japan) were saccharified with the cellulases, 76.8% of the substrate was converted into glucose within 5 h, whereas the corresponding conversion rate of water-pretreated cellulose flakes was 33.8%. To clarify the mechanism of the promotion, cellobiohydrolase I purified from Trichoderma longibrachiatum was used as the model cellulase, which degraded CaCl2-pretreated cellulose more quickly than the water-pretreated cellulose under tested conditions. The maximum amount of the enzyme bound to CaCl2-pretreated cellulose at 37 degrees C was estimated as 1.14 nmol/mg of cellulose, whereas that to water-pretreated cellulose was 0.527 nmol/mg of cellulose. The specific activity of the bound enzyme greatly decreased with the increase of the surface density (rho) of the bound enzyme, and no significant positive effects of the CaCl2-pretreatment on the specific activity could be observed at the same rho value, suggesting that the promotion was attributed mainly to the increase of the surface area of cellulose. The effect was also observed with dewaxed cotton or filter paper, but not with nata de coco cellulose or bagasse cellulose as the substrates. This suggests that the CaCl2-pretreatment serves to increase the surface area of cellulose flakes via liberation of cellulose particles which were artificially aggregated during harsh drying processes of the flakes.     

Purification and characterization of a chitinase from Amycolatopsis orientalis with N-acetyllactosamine-repeating unit releasing activity

We report a novel enzyme from the culture filtrate of Amycolatopsis orientalis, that endoglycosidically releases an N-acetyllactosamine-repeating unit (Galbeta1,4GlcNAcbeta1,3Galbeta1,4GlcNAc, LN2) from a synthetic chromogenic substrate Galbeta1,4GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (1). The enzyme activity was purified by 80% saturated ammonium sulfate precipitation followed by gel filtration and affinity chromatography. The enzyme splits 1, Galbeta1,4GlcNAcbeta-pNP (2), GlcNAcbeta1,3Galbeta1,4GlcNAcbeta-pNP (3), and GlcNAcbeta1,4GlcNAcbeta-pNP (4) into the corresponding oligosaccharides and p-nitrophenol. The catalytic efficiencies (k(cat)/K(m)) for compounds 1, 2, and 4 were 0.6, 0.05, and 13, respectively. Compound 4 acts as a fairly good substrate for the enzyme, and LN2-releasing activity was inhibited by 4 and GlcNAcbeta1,4GlcNAcbeta1,4GlcNAcbeta-pNP (7), indicating that this enzyme activity is derived from a kind of chitinase. The enzyme hydrolyzed 1 by a mechanism leading to retention of the anomeric configuration. This is the first report of a N-acetyllactosamine-repeating unit releasing enzyme.     

A scaffoldless technique for self-generation of three-dimensional keratinospheroids on liquid crystal surfaces

We describe a new scaffold-free three-dimensional (3D) cell culture model using cholesteryl ester based lyotropic liquid crystal (LC) substrates. Keratinocytes were deposited randomly on the LC surface where they self-assembled into 3D microtissues or keratinospheroids. The cell density required to form spheroids was optimized. We investigated cell viability using dead/live cell assays. The adhesion characteristics of cells within the microtissues were determined using histological sectioning and immunofluorescence staining. Fourier transform infrared spectroscopy (FTIR) was used to characterize the biochemistry of the keratinospheroids. We found that both cells and microtissues could migrate on the LC surface. The viability study indicated approximately 80% viability of cells in the microtissues up to 20 days of culture. Strong intercellular adhesion was observed in the stratification of the multi-layered microspheroids using field emission-scanning electron microscopy (FE-SEM) and histochemical staining. The cytoskeleton and vinculins of the cells in the microtissues were expressed diffusely, but the microtissues were enriched with lipids and nucleic acids, which indicates close resemblance to the conditions in vivo. The basic 3D culture model based on LC may be used for cell and microtissue migration studies in response to cytochemical treatment.     

Veränderung der Melaninmenge beim Farbwechsel der Fische Esox,Carassius, Phoxinus; Gobius; Nemachilus

Ohne ZusammenfassungEin Auszug dieser Arbeit erschien mit gleichlautendem Titel als Mitteilung Nr. 61 aus der Biolog. Versuchsanstalt der Akademie der Wissenschaften, Zoologische Abteilung, Vorstand H. Przibram, im Akadem. Sitzungsanzeiger, Wien, Nr. 14, 1921.     

Localization of Cytochrome b-559 in the Chloroplast Thylakoid Membranes in Spinach

Cytochrome b-559 was purified from spinach leaves and antibodies were made against it in rabbit. Using affinity-purified, monospecific antibodies, we have found that cytochrome b-559, which is closely associated with the primary photochemical activity of photosystem II, is localized exclusively in the grana thylakoids.