Antibodies raised against denatured DNA bind to double-stranded DNA

Antibodies raised against denatured DNA complexed with methylated bovine serum albumin have been reported to react with ssDNA but not with dsDNA. Using a highly sensitive avidin-biotin microELISA, we report that such antibodies also bind to dsDNA. Antibodies which reacted with ssDNA and dsDNA were found to be IgG type. The antibodies did not react with tRNA and rRNA. The binding of antibodies to dsDNA was partially inhibited dy individual deoxyribonucleotides. ssDNA as well as dsDNA inhibited the binding of antibodies to dsDNA. The binding of these antibodies to supercoiled and relaxed forms of pBR322 DNA was demonstrated by gel retardation assay. The cross-reaction with ssDNA was observed even after affinity purification on native DNA-cellulose. The antibodies were also shown to bind to poly(dA-dT)·poly(dA-dT)

Expression of drug resistance markers by spheroplasts ofMycobacterium smegmatis after fusion

Mycobacterial spheroplasts were prepared by treatment of the glycinesensitized cells with a combination of lipase and lysozyme. They were stable for several hours at room temperature but were lysed on treatment with 0.1% sodium dodecyl sulfate. The spheroplasts could be regenerated on a suitable medium. Fusion and regeneration of the spheroplasts were attempted using drug resistant mutant strains ofM. smegmalis. Recombinants were obtained from spheroplast fusion mediated by polyethylene glycol and dimethyl sulfoxide. Simultaneous expression of rccombinant properties was observed only after an initial lag in the isolated clones. This has been explained as due to “chromosome inactivation” in the fused product.

Purification and characterization of a beta-glucosidase and endocellulase from Humicola insolens

A .beta.-glucosidase and an endocellulase were purified from the culture filtrates of a thermophilic cellulolytic fungus Humicola insolens. Both the preparations were homogeneous by PAGE, ultracentrifugation and gel filtration (Mr 45,000). Ouchterlony immunodiffusion showed complete cross reactivity between the antibodies and the two enzyme antigens, indicating the presence of a common epitope on the two enzyme proteins. The two enzymes, however, differ in their amino acid composition and their substrate specificity. .beta.-Glucosidase acts on p-nitrophenyl .beta.-D-glucopyranoside and hydrolyses cellulose to release mainly glucose and small amounts of cellobiose from the non-reducing end. On the other hand, endocellulase hydrolyses cellulose to release cellopentaose, cellotetraose, cellotriose along with cellobiose and glucose and also hydrolyses larch wood xylan.

Purification of goat serum retinol-binding protein and preparation of its antibodies

The method for the purification of goat serum retinol-binding protein consists of DEAE-cellulose chromatography of the serum followed by preparative polyacrylamide disc gel electrophoresis. After electrophoresis, the retinol-binding protein containing zone is identified by the specific fluorescence of retinol. For raising the antibodies, the portion of the gel containing retinol binding protein is homogenized and injected intradermally and intramuscularly to rabbits. The availability of this simple method for the isolation of retinol-binding protein and production of its antibodies enables the development of a radioimmunoassay for this protein.

Studies on LDPE-cyanoethylated lignocellulosics blends using epoxy functionalized LDPE as compatibilizer

Rubberwood flour and cellulose have been plasticized by cyanoethylation and then blended with low-density polyethylene (LDPE). A small quantity of epoxy functionalized polyethylene i.e., polyethylene-co-glycidyl methacrylate (PEGMA) has been added to further enhance the mechanical properties. The mechanical properties were measured according to the standard ASTM methods. SEM analysis was performed for both fractured and unfractured blend specimens. The mechanical properties were improved by the addition of PEGMA compatibilizer. LDPE blends with cyanoethylated wood flour (CYWF) showed higher tensile strength and modulus than cyanoethylated cellulose CYC-LDPE blends. However CYC-LDPE blends exhibited higher relative elongation at break values as compared with the former. The TGA analysis showed lowering of thermal stability as the filler content is increased and degradation temperature of LDPE is shifted slightly to lower temperature. DSC analysis showed loss of crystallinity for the LDPE phase as the filler content is increased for both types of blends. Dielectric properties of the blends were similar to LDPE, but were lowered on adding PEGMA. (c) 2006 Wiley Periodicals, Inc.

Nanoscale studies of materials using x-ray synchrotron radiation and mesoscopic modelling

X-ray synchrotron radiation was used to study the nanostructure of cellulose in Norway spruce stem wood and powders of cobalt nanoparticles in cellulose support. Furthermore, the growth of metallic clusters was modelled and simulated in the mesoscopic size scale. Norway spruce was characterized with x-ray microanalysis at beamline ID18F of the European Synchrotron Radiation Facility in Grenoble. The average dimensions and the orientation of cellulose crystallites was determined using x-ray microdiffraction. In addition, the nutrient element content was determined using x-ray fluorescence spectroscopy. Diffraction patterns and fluorescence spectra were simultaneously acquired. Cobalt nanoparticles in cellulose support were characterized with x-ray absorption spectroscopy at beamline X1 of the Deutsches Elektronen-Synchrotron in Hamburg, complemented by home lab experiments including x-ray diffraction, electron microscopy and measurement of magnetic properties with a vibrating sample magnetometer. Extended x-ray absorption fine structure spectroscopy (EXAFS) and x-ray diffraction were used to solve the atomic arrangement of the cobalt nanoparticles. Scanning- and transmission electron microscopy were used to image the surfaces of the cellulose fibrils, where the growth of nanoparticles takes place. The EXAFS experiment was complemented by computational coordination number calculations on ideal spherical nanocrystals. The growth process of metallic nanoclusters on cellulose matrix is assumed to be rather complicated, affected not only by the properties of the clusters themselves, but essentially depending on the cluster-fiber interfaces as well as the morphology of the fiber surfaces. The final favored average size for nanoclusters, if such exists, is most probably a consequence of these two competing tendencies towards size selection, one governed by pore sizes, the other by the cluster properties. In this thesis, a mesoscopic model for the growth of metallic nanoclusters on porous cellulose fiber (or inorganic) surfaces is developed. The first step in modelling was to evaluate the special case of how the growth proceeds on flat or wedged surfaces.

Litter quality and its response to water level drawdown in boreal peatlands at plant species and community level

Changes in the structure of plant communities may have much more impact on ecosystem carbon (C) cycling than any phenotypic responses to environmental changes. We studied these impacts via the response of plant litter quality, at the level of species and community, to persistent water-level (WL) drawdown in peatlands. We studied three sites with different nutrient regimes, and water-level manipulations at two time scales. The parameters used to characterize litter quality included extractable substances, cellulose, holocellulose, composition of hemicellulose (neutral sugars, uronic acids), Klason lignin, CuO oxidation phenolic products, and concentrations of C and several nutrients. The litters formed four chemically distinct groups: non-graminoid foliar litters, graminoids, mosses and woody litters. Direct effects of WL drawdown on litter quality at the species level were overruled by indirect effects via changes in litter type composition. The pristine conditions were characterized by Sphagnum moss and graminoid litters. Short-term (years) responses of the litter inputs to WL drawdown were small. In longterm (decades), total litter inputs increased, due to increased tree litter inputs. Simultaneously, the litter type composition and its chemical quality at the community level greatly changed. The changes that we documented will strongly affect soil properties and C cycle of peatlands.

A simplified method for the purification of riboflavin-binding protein from hen's egg yolk

A simple and efficient procedure for the purification of the riboflavin-binding protein from hen's egg yolk is described. This method involves the removal by exclusion of lipoproteins and subsequent fractionation of soluble yolk proteins held on a DEAE-cellulose column by a salt gradient which is followed by purification by gel filtration on Sephadex G-100. The protein thus isolated is homogeneous by various physicoehemical, immunological, and functional criteria.

Selective sorption of ferric ion onto a crosslinked resin bearing triphenylphosphinimine residues

A polymeric sorbent containing triphenylphosphinimine residues has been obtained from crosslinked chloromethylated polystyrene by azidation, using phase-transfer catalysis, followed by reaction with triphenylphosphine at room temperature. The sorbent exhibits 100 % sorption selectivity for Fe(III) in the presence of Cu(II), Fe(II), Ni(II), Co(II), Mn(II), and Zn(II) in aqueous media. In the absence of Fe(III), however, Fe(II) is selectively sorbed over the other metal ions, and in the absence of both Fe(II) and Fe(III), Cu(II) has the highest selectivity of sorption on the resin. The sorption of Fe(III) is sensitive to pH, being maximum at pH not, vert, similar 2 and falling sharply at both higher and lower pH values. The sorbed Fe(III) is easily stripped with dilute HCl and the resulting protonated resin is regenerated to its original sorption capacity by treatment with dilute NaOH at room temperature.

The effects of beta-mercaptoethanol and sodium dodecyl sulfate on the Humicola insolens beta-glucosidase

Hydrolysis of p-nitrophenyl-beta-D-glucoside by the beta-glucosidase of a thermophilic and cellulolytic fungus, Humicola insolens was stimulated by two-fold in the presence of high concentrations of beta-mercaptoethanol. This enzyme did not have any free sulfhydryl groups and high concentrations of beta-mercaptoethanol (5% v/v) reduced all of the three disulfide bonds present in the enzyme. In contrast, the hydrolysis of cellobiose and cellulose polymers was inhibited by 50% under the same conditions. Sodium dodecyl sulfate (1% w/v) even in combination with beta-mercaptoethanol did not show any significant effects on this enzyme. These unusual properties suggest that this enzyme may be of significant importance for understanding the structure of the enzyme.

Development of tissue culture and virus-induced gene silencing for Calendula officinalis

Calendula officinalis is grown widely as an ornamental plant across Europe. It belongs to the large. Asteraceae family. In this study, the aim was to explore the possibilities to use Calendula officinalis as a new model organism for flower development and secondary mechanism studies in Asteraceae. Tissue culture of Calendula officinalis was established using nine different cultivars. Murashige & Skoog (MS) medium with four different combinations of plant growth regulators were tested. Of all these combinations, the medium containing 1mg/l BAP, 0.1 mg/l IAA, and 1mg/l Zeatin achieved highest frequency of adventitious shoot regeneration from hypocotyl and cotyledon explants. Virus-induced gene silencing is a recent developed genetic tool for charactering the gene functions in plants, and extends the range of host plants that are not accessible for Agrobacterium transformation. Here, tobacco rattle virus (TRV)-based VIGS technique was tested in calendula (cv. Single Orange). We used TRV carrying Gerbera hybrid phytoene desaturase (PDS) gene fragment to induce PDS silencing in calendula. Vacuum infiltration and syringe infiltration methods both resulted in photo-bleaching phenotypes in leaves, bracts and petals. Loss-of-function phenotypes occurred on calendula 13 days post-infiltration. In conclusion, the data indicates that calendula explants can be regenerated through tissue culture which is a prerequisite for development of stable transformation methods. However, further optimization is still needed to improve the frequency. In addition, VIGS was applied to silence PDS marker gene expression indicating that this method has potential for gene functional studies in future.

Purification and functional characteristics of an endocellulase from Chaetomium thermophile var. coprophile

An endocellulase (1→4)-β-d-glucan 4-glucanohydrolase was isolated from the culture filtrates of Chaetomium thermophile. The enzyme was homogeneous by PAGE and SDS-PAGE. The molecular weight was 36 000 by SDS-PAGE and 38 000 by gel filtration. It was a glycoprotein. From the amino acid composition, it was found to be rich in glycine, threonine, and aspartic and glutamic acids, but contained only low proportions of histidine and sulfur-containing amino acids. It was optimally active at pH 6 and at 60°. The enzyme did not hydrolyze cellobiose and cellotriose, but hydrolyzed cello-tetraose, -pentaose, and -hexaose at comparable rates. It was specific for molecules containing β-(1→4) linkages. It showed high activity towards amorphous cellulose, and the reaction products contained cellobiose to cellopentaose, showing that it effects random cleavage of cellulose.

Purification and characterization of an extracellular β-glucosidase from the thermophilic fungus Sporotrichum thermophile and its influence on cellulase activity

Multiple forms of beta-glucosidase (EC 3.2.1.21) of Sporotrichum thermophile were produced when the fungus was grown in a cellulose medium. One beta-glucosidase was purified 16-fold from 6-d-old culture filtrates by ion-exchange and gel-filtration chromatography. The purified enzyme was free of cellulase activity. It hydrolysed aryl beta-D-glucosides and beta-D-linked diglucosides. It was optimally active at pH 5.4, at 65-degrees-C. The apparent K(m) values for p-nitrophenyl beta-D-glucoside (PNPG) and cellobiose were 0.29 and 0.83 mm, respectively. Glucose, fucose, nojirimycin and gluconolactone inhibited beta-glucosidase competitively. At high (> 1 mm) substrate concentration, beta-glucosidase catalysed a parallel transglycosylation reaction. The transglycosylation product formed from cellobiose appeared to be a beta-linked tetramer of glucose. Admixtures of beta-glucosidase and cellulase components showed that the concept of cellobiose inhibition of cellulases was not valid for all components of the cellulase system of S. thermophile. Beta-Glucosidase supplementation also stimulated cellulose hydrolysis by cellulases when there was no accumulation of cellobiose in reaction mixture.

Regeneration of plantlets from leaf disc cultures of rosewood: control of leaf abscission and shoot tip necrosis

A method for mass production of rosewood (Dalbergia latifolia Roxb.) trees through leaf disc organogenesis was developed and standardized. Compact callus was initiated from mature leaf discs on Murashige and Skoog (MS) basal medium supplemented with 1.0 mg 1?1 2,4-dichlorophenoxy acetic acid (2,4-D), 5.0 mg 1?1 ?-naphthaleneacetic acid (NAA), 1.0 mg 1?1 6-benzylaminopurine (BAP) and 10% coconut water (CW). High frequency (15�20 shoots/g callus) regeneration of shoot bud differentiation was obtained on MS (3/4 reduced major elements) or Woody Plant Medium (WPM) or modified Woody Plant Medium (mWPM) supplemented with BAP (5.0 mg 1?1) and NAA (0.5 mg 1?1). Leaf abscission and shoot tip necrosis was controlled using mWPM. About 90% of the excised shoots were rooted in the mWPM supplemented with 2.0 mg 1?1 ?-indolebutyric acid (IBA) and 1.0 mg 1?1 caffeic acid. The in vitro-raised rooted plantlets were hardened for successful transplantation to soil. The transplanted plants were exposed to various humidity conditions and 80% transplant success was achieved. The in vitro-raised leaf-regenerated plants grew normally and vigorously in soil.

Localization and release of ?-glucosidase in the thermophilic and cellulolytic fungus, Sporotrichum thermophile

In a medium containing cellulose as the carbon source, the rapid growth of Sporotrichum thermophile, the secretion of cellulases and the utilization of cellulose were well-correlated events. The production of beta-glucosidase in culture medium lagged behind cellulases, coinciding with the time of extensive autolysis of mycelia. By contrast, neither apparent autolysis nor secretion of beta-glucosidase occurred when S. thermophile was grown in medium containing cellobiose; the enzyme activity remained associated with mycelia. The release of beta-glucosidase in cellulose-grown cultures was correlated with the activity of the lytic enzyme in the cell wall. Immunocytochemical localization and biochemical characterization showed that a beta-glucosidase released in the cellulose medium was the same as that which remained associated with mycelia grown on cellobiose. The results indicated that the release of beta-glucosidase in the cellulose culture is incidental to the activity of the lytic enzymes which are strongly induced by cellulose. The observations minimize a functional role of the culture fluid beta-glucosidase in cellulolysis by the fungus. Rather, the available information suggests that the cellulases and beta-glucosidases associated with the hyphal cell wall may play a role in cellulolysis by the fungus. (C) 1994 Academic Press, Inc.