Immunogold-cytochemical labelling of β-glucosidase in the white-rot fungus Coriolus versicolor

Summary Immunogold cytochemical labelling of hyphal sections of Coriolus versicolor showed that -glucosidase was localised in the extracellular mucilage, cell wall layers and cell interior in hyphae grown on glucose-rich malt extract medium whereas in hyphae grown with carboxymethylcellulose (CMC) as sole carbon source, most labelling was in the cell wall layers and cell interior. Little mucilage was visible around hyphae from these cultures. Hyphae from beechwood cultures showed gold labelling of -glucosidase in mucilage and fungal cell walls with some intracellular labelling. Biochemical studies of enzyme activity showed that similar amounts of enzyme were detected in the growth medium when cultures were grown on CMC medium, in agitated liquid cultures or in stationary cultures. In agitated cultures grown on glucose-rich malt extract, the activity of -glucosidase in the medium was 100 times less than that detected in stationary cultures on the same medium. However activity in the hyphae of stationary CMC-grown cultures was similar to that in hyphae from stationary glucose-rich cultures. These data confirm the patterns of gold labelling observed in hyphae from stationary cultures on glucose-rich malt extract when -glucosidase was immobilised in the extracellular mucilage layer around the hyphae. In this paper we propose that a primary function of the extracellular mucilage produced by hyphae of C. versicolor in vivo is to serve as a matrix for immobilisation of -glucosidase. Its substrate, cellobiose, which is released as a result of endo-and exoglucanase hydrolysis of cellulose, is absorbed and retained by the gel filtration properties of the mucilage, so encountering the immobilised -glucosidase. Glucose produced by this reaction is retained within the mucilage matrix around the hyphae before intracellular absorption.Offprint requests to: C. S. Evans     

Growth inhibition of selected food-borne bacteria, particularly Listeria monocytogenes, by plant extracts

C hung , K.-T., T homasson , W.R. & W u -Y uan , C.D. 1990. Growth inhibition of selected food-borne bacteria, particularly Listeria monocytogenes , by plant extracts. Journal of Applied Bacteriology 69 , 498–503.
Six extracts from Chinese medicinal plants: Tin Men Chu, Sey Lau Pai, Siu Mao Heung, Bak Tao Yung, Kam Chin Chiu and Liao Ya, were tested for their inhibitory effect on selected food-borne bacteria by the well assay technique. Among them, Tin Men Chu, Siu Mao Heung and Sey Lau Pai inhibited the growth of Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli, Shigella flexneri, Streptococcus faecalis, Salmonella paratyphi, Salm. enteritidis, Enterobacter aero-genes, Pseudomonas fluorescens, Proteus vulgaris, Alcaligenes faecalis , and three strains of Listeria monocytogenes . Two of these three extracts, Tin Men Chu and Siu Mao Heung, suppressed the growth of L. monocytogenes Scott A in cabbage juice. This inhibition was prevented by the addition of protein but not sodium chloride. Plant extracts show potential to control the growth of food-borne bacteria.     

Duplication of the PMP22 gene in 17p partial trisomy patients with Charcot-Marie-Tooth type-1A neuropathy

Autosomal dominant Charcot-Marie-Tooth type-1A neuropathy (CMT1A) is a demyelinating peripheral nerve disorder that is commonly associated with a submicroscopic tandem DNA duplication of a 1.5-Mb region of 17p11.2p12 that contains the peripheral myelin gene PMP22. Clinical features of CMT1A include progressive distal muscle atrophy and weakness, foot and hand deformities, gait abnormalities, absent reflexes, and the completely penetrant electrophysiologic phenotype of symmetric reductions in motor nerve conduction velocities (NCVs). Molecular and fluorescence in situ hybridization (FISH) analyses were performed to determine the duplication status of the PMP22 gene in four patients with rare cytogenetic duplications of 17p. Neuropathologic features of CMT1A were seen in two of these four patients, in addition to the complex phenotype associated with 17p partial trisomy. Our findings show that the CMT1A phenotype of reduced NCV is specifically associated with PMP22 gene duplication, thus providing further support for the PMP22 gene dosage mechanism for CMT1A. Received: 3 May 1995 / Revised: 1 August 1995     

Kanamycin rescue: A simple technique for the recovery of T-DNA flanking sequences

We have designed a new method for the recovery of T-DNA flanking sequences from T-DNA-tagged lines ofArabidopsis thaliana. Since most transformation vectors in use contain a plant-selectable marker for kanamycin resistance, we can use the 3′ part of thenptII coding region from the T-DNA to complement the bacterial 5′ region of thenptII gene from Tn5 to reconstruct a functional kanamycin-resistance gene inEscherichia coli. We have constructed a vector that contains the 5′ part of thenptII gene from Tn5 up to the uniquePst I site. By cloning total DNA from transformed lines in this vector, we were able to select directly for clones containing a T-DNA fragment, which reconstitutes a functional kanamycin gene, and a fragment of arabidopsis genomic DNA adjacent to the insertion. Flanking sequences up to 4 kb were rescued by this system.     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

Quantitative floral development in Pseudolysimachion (Scrophulariaceae): intraspecific variation and comparison with Veronica and Veronicastrum

Intraspecific variation in floral development was studied in four morphologically distinct strains of Pseudolysimachion longifolium and two of P. spicatum. Size increase of whole buds from living plants of P. longifolium was followed in absolute time. Growth rates were homogeneous within inflorescences and within individual plants, and significantly different between individual plants. Organ growth trajectories (plotted as organ size over gynoecium size) differed little among individuals of Pseudolysimachion belonging to morphologically distinct strains; variation was most pronounced in the calyx. Corolla tube as well as calyx and corolla lobe measurements varied more during the early stages of development than near maturity, suggesting constraints on mature flower form. In addition to intraspecific variation, floral development in Pseudolysimachion was compared with that in related Veronica chamaedrys and Veronicastrum virginicum, using data from a previous study. Mature flowers of Pseudolysimachion have long corolla tubes similar to those of Veronicastrum and long corolla lobes similar to those of Veronica. The trajectories of most organs of Pseudolysimachion were intermediate between those of Veronica and Veronicastrum in position as well as in type of curve, i.e., different trajectories lead to similar end products; thus, the shapes of the mature organs are not strictly homologous and not indicative of close relationship.