Bacteriophage Ø105clz induces the GroEL-homologue protein inBacillus subtilis

Using two-dimensional polyacrylamide gel electrophoresis, the GroEL homologue ofBacillus subtilis was shown to be induced upon infection with Ø105clz, a clear plaque mutant of the temperate bacteriophage Ø105. Western blotting of one dimensional polyacrylamide gels also showed the induction of the GroEL homologue when cells were infected with Ø105clz.     

Elimination of apple stem grooving virus by chemotherapy and development of an immunocapture RT-PCR for rapid sensitive screening

Ombuin (7,4′-dimethyl quercetin) (10 μg ml^-1, for 12 wk), glycyrrhizin/quercetin (80 μg ml^-1and 10 μg ml^-1respectively, for 18 wk), ribavirin (10 μg ml^-1, for 12 wk) and quercetin/ribavirin (10 μg ml^-1each, for 9–12 wk) reduced the titre of apple stem grooving virus (ASGV) when applied in vitro to infected tissue cultures of Nicotiana occidentalis obliqua Wheeler, and/or Malus domestica. ASGV was not detectable in both plant species after the quercitin/ribavirin treatment when tested by ISEM, herbaceous host indexing, RT-PCR, and immunocapture RT-PCR. A sensitive immunocapture RT-PCR procedure for the detection of ASGV was developed for the screening of treated samples to assess antiviral activity.     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.     

An Inhibitor of the UDP-N-Acetylgalactosamine:GM3, N-Acetylgalactosaminyltransferase: Purification and Properties, and Preparation of an Antibody to This Inhibitor

An inhibitor of the UDP-N-acetylgalactosamine:GM3, N-acetylgalactosaminyltransferase (EC 2.4.1.92) has been purified close to 100-fold from chicken blood serum. The method of purification includes heating, dialysis, passage through a column of DEAE-Sephadex, filtration through Amicon XM 100, and passage through Sepharose 6B. The molecular weight determined by Sepharose 6B was 200,000, but on sodium dodecyl sulfate-polyacrylamide gel electrophoresis it appears as if the compound dissociated into components of 68,000. The inhibitor was not active on other glycosyl transferases and lost its inhibitory activity following treatment with pronase and trypsin. alpha-Chymotrypsin did not affect the inhibitor. An antibody to this inhibitor was prepared which decreased its inhibitory capability and precipitated with it in a radial double immunodiffusion experiment.     

Cloning in Escherichia coli of genes involved in the synthesis of proline and leucine in Desulfovibrio desulfuricans Norway

Summary A library of Deusulfovibrio desulfuricans Norway genomic DNA was constructed in Escherichia coli with pBR322 as vector and plasmids able to complement the proA and leuB mutations of the host were screened. It was observed that all the plasmids studied were highly unstable, the insert DNA being rapidely lost under non-selective growth conditions. A 2.75 kb DNA fragment of D. desulfuricans Norway was found to complement E. coli ProA, ProB and ProC deficiencies. From the results of restriction analysis and Southern hybridization, it is proposed that the genes involved in proline and leucine biosynthesis are clustered on the chromosome of D. desulfuricans Norway.     

Immuno-gold localization of α-L-arabinofuranosyl residues in pollen tubes of Nicotiana alata Link et otto

Immuno-gold labelling using a monoclonal antibody (PCBC3) with a primary specificity for -L-arabinofuranosyl residues was used to locate these residues in pollen tubes of Nicotiana alata grown in vivo. The antibody bound to the outer fibrillar layer of the pollen-tube wall: the inner, non-fibrillar wall layer was not labelled. Cytoplasmic vesicles (0.2 m diameter) were also labelled. The antibody may bind to an arabinan in the pollen-tube wall.