Direct gene transfer into Actinidia deliciosa protoplasts: analysis of transient expression of the CAT gene using TLC autoradiography and a GC-MS-based method

Chloramphenicol acetyl transferase (CAT) gene was used as a reporter gene to assess the conditions for polyethylene glycol (PEG)-mediated transfection of kiwifruit protoplasts. The effect of plasmid concentration and the presence of carrier DNA were each assessed by analysing CAT activity in transfected protoplasts using thin-layer chromatography (TLC) autoradiographic detection of acetylated chloramphenicol. A gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) non-radioactive method was developed for monitoring CAT gene activity. This method provides a high speed of analysis (30 min) and precise means of detecting acetylated products at the nanomolar level, enabling quantification at very low transfection rates. Using this method we optimized plasmid and PEG concentration and also assessed the effect of heat shock on transfection. The best CAT activity was obtained using 30% polyethylene glycol 4000 and by submitting protoplasts to heat shock (45 °C, 5 min) prior to transfection.     

Hidden smectic properties of a chiral side-chain co-oligomer

We recently showed that a side-chain industrial co-oligosiloxane presents a quenchable enlarged blue phase behaviour at the cholesteric-isotropic phase transition. In this paper, we present the results of a structural study based on X-ray diffraction, differential scanning calorimetry and optical measurements. In particular, the smectic A organisation is demonstrated in the lower temperature domain, which was hitherto understood as a cholesteric phase. A structural model for this phase is proposed on the basis of the analysis of the anisotropic scattering of stretched fibers. Our results also suggest that the observed glass transition is indeed a rather complex phenomenon, which seems to involve not only the freezing of the main chains, but also smectic correlations at the side-chain level. Moreover, the calorimetric study indicates that, notwithstanding the conservation of the processed film's optical properties, low kinetic reorganisations occur at room temperature.     

Biotransformation of 2-acetylthiophene by micromycetes

Summary The biotransformation of 2-acetylthiophene by 800 strains of micromycetes has been investigated. Among them, 460 strains have been selected on solid media and cultivated in a liquid synthetic medium. Of these, 106 strains were able to completely deplete 2-acetylthiophene with or without production of intermediary metabolites. 2-Thienylglyoxylic acid was not detected but 72 strains produced 2-thiophenecarboxylic acid. Among them, eight strains have been selected for further optimization of this bioconversion.     

Action of Bacillus subtilis alpha-amylase on native wheat starch

Native starch granules from wheat have been subjected to enzymatic depolymerization with an alpha-amylase from Bacillus subtilis. Crystallites made from short-chain amylose and residues from mild acid hydrolysis have been also tested. Electron microscopy, particle size analysis, DSC, and x-ray diffractometry reveal that enzymatic degradation occurs granule by granule. Gel permeation chromatography shows off the macromolecular nature of the remaining material. In contrast, acid erodes simultaneously all the granules, leading to a splitting into small particles. Crystalline fractions are completely degraded by alpha-amylase. These results support evidence for an active disentanglement of chains, carried out by the different subsites of alpha-amylase molecules. A simple mathematical treatment is proposed to explain the results of the kinetics.     

Modeling dynamic experiments on the anaerobic degradation of molasses wastewater

The kinetics of anaerobic degradation of a molasses wastewater were measured under constant pH conditions in a laboratory scale packed bed reactor. In continuous and batch experiments the formation and degradation rates of the organic acids (butyric, propionic and acetic) have been followed. The influence of hydrogen gas on the acid degradation rates has been measured and, contrary to the literature and the thermo-dynamic calculations, no inhibition was detected, biofilm diffusional effects may be the reason. Two dynamic simulation models were tested, a heterogeneous model, which considered the biofilm diffusion-reaction phenomena and a quasihomogeneous model with the same kinetics. Except for hydrogen, the diffusion effects were found to be negligible. Otherwise both models gave essentially the same results and the time profiles of acids, hydrogen, carbon dioxide and methane agreed relatively well with dynamic startup experiments. Batch experiments showed the acid concentrations to be highly sensitive to the initial molasses concentration. This aspect was not included in the model but is being investigated further.     

Tolerance induction by a poorly arthritogenic collagen II can prevent collagen-induced arthritis

Collagen type II (CII)-induced arthritis (CIA) can be induced in 78% of B10.RIII mice (H2r) by intradermal (id) immunization with CII of bovine origin in complete Freund's adjuvant (CFA), whereas immunization with CII of chick origin induces arthritis in less than 5% of these mice. Nevertheless, tolerization of B10.RIII mice with intravenously injected chick CII renders the animals resistant to induction of CIA by immunization with bovine CII. Such tolerization can be achieved either by intravenous injection of 500 micrograms chick CII 1 week prior to immunization with bovine CII in CFA or by such an intravenous injection of chick CII 2 weeks after immunization with bovine CII in CFA. Postimmunization treatment results in a significant decrease in the concentration of antibody to bovine CII. Preimmunization administration of chick CII causes a marked decrease in the antibody reactive with chick CII without a significant effect on the anti-bovine CII antibody concentration. In DBA/1 mice, a strain in which both bovine CII and chick CII can induce a high incidence of the disease, intravenous injection of bovine CII can also prevent arthritis induced by chick CII, even when given 7 or 14 days after immunization. The fact that chick CII as tolerogen is quite effective in preventing arthritis in B10.RIII mice, while as immunogen it is very ineffective in inducing arthritis in this strain, may be interpreted as evidence for interaction between different epitopes on CII in the pathogenesis of CIA.     

Pigment patterns in mutants affecting the biosynthesis of pteridines and xanthommatin in Drosophila melanogaster

Eye-color mutants of Drosophila melanogaster have been analyzed for their pigment content and related metabolites. Xanthommatin and dihydroxanthommatin (pigments causing brown eye color) were measured after selective extraction in acidified butanol. Pteridines (pigments causing red eye color) were quantitated after separation of 28 spots by thin-layer chromatography, most of which are pteridines and a few of which are fluorescent metabolites from the xanthommatin pathway. Pigment patterns have been studied in 45 loci. The pteridine pathway ramifies into two double branches giving rise to isoxanthopterin, drosopterins, and biopterin as final products. The regulatory relationship among the branches and the metabolic blockage of the mutants are discussed. The Hn locus is proposed to regulate pteridine synthesis in a step between pyruvoyltetrahydropterin and dihydropterin. The results also indicate that the synthesis and accumulation of xanthommatin in the eyes might be related to the synthesis of pteridines.Support for this work was provided to J.F. in part by a grant from the Ministerio de Universidades e Investigación (Spain) and to F.J.S. by a grant from the Ministerio de Educación y Ciencia (Spain).     

Molecular cloning and characterization of the trpC gene from Penicillium chrysogenum

Summary We cloned the Penicillium chrysogenum trpC gene from a genomic library by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase activity. The gene ecodes a 2.7 kb poly(A)⁺ RNA. We localized the gene by sequence analysis in a 2.9 kb DNA insert found in the smallest plasmid selected from the library. Sequence data strongly suggest that the organization of the gene is similar to that described in other Ascomycetes. We found that a DNA fragment which codes only for the carboxy-terminal protion of the polypeptide is sufficient for complementation of the E. coli trpC9830 mutation.     

Membrane Operational Impedance Spectra in Chara corallina Estimated by Laplace Transforms Analysis

The membrane operational impedance spectrum of Chara corallina Klein ex Willd. (R. Brown) cells is investigated using Laplace transform analysis. The spectrum changes with both amplitude and sign of the electrical stimulation when time- and voltage-dependent K⁺ channels contribute to the membrane conductance. We compare the advantages and disadvantage of this technique for studying membrane impedance with those of the alternating current method and the white noise method.