Systemic Scedosporium prolificans infection in an 11-month-old Border collie with cobalamin deficiency secondary to selective cobalamin malabsorption (canine Imerslund-Gräsbeck syndrome).

An 11-month-old Border collie presented collapsed and continued to deteriorate rapidly despite supportive treatment. The dog had a history of failure to thrive and recurring respiratory infection. Laboratory abnormalities included neutrophilic leucocytosis, Heinz body anaemia, hyperammonaemia, hyperbilirubinaemia, proteinuria and hypocobalaminaemia. Post-mortem examination revealed multi-focal necrosis within the heart, kidneys, pancreas, liver, meninges and cerebral cortex. Fungal hyphae in lesions were identified as Scedosporium prolificans following culture. Subsequent genotyping confirmed that the dog carried the CUBN:c.8392delC mutation in a homozygous state, verifying hereditary cobalamin deficiency (a.k.a. Imerslund-Gräsbeck syndrome). Cobalamin deficiency may have been a predisposing factor for the development of systemic fungal infection in this dog.

Clinical findings and results of diagnostic imaging in 82 dogs with gastrointestinal ulceration.

OBJECTIVES: To describe clinical and imaging findings in dogs with confirmed gastrointestinal ulceration, to compare findings in dogs with perforated and non-perforated ulcers and to estimate the sensitivities of radiography, ultrasonography and computed tomography (CT) for gastrointestinal ulceration and perforation. METHODS: Retrospective review of medical records of 82 dogs that had a macroscopic ulcer in the gastric or intestinal mucosa diagnosed directly at endoscopy, surgery or necropsy and had survey radiography, ultrasonography or a CT scan of the abdomen during the same period of hospitalisation. RESULTS: The most frequent clinical signs were vomiting in 88% dogs, haematemesis in 32%, melaena in 31% and weight loss in 7%. The most frequent imaging findings in dogs with non-perforated ulcers were gastrointestinal mural lesion in 56%, mucosal defect compatible with an ulcer in 44% and peritoneal fluid in 21%. In dogs with perforated ulcers the most frequent imaging findings were peritoneal fluid in 83%, gastrointestinal mural lesion in 48%, peritoneal gas in 31% and mucosal defect compatible with an ulcer in 29%. Sensitivities of radiography, ultrasonography and CT were 30, 65 and 67% in dogs with non-perforated ulcers and 79, 86 and 93% in dogs with perforated ulcers, respectively. CLINICAL SIGNIFICANCE: In dogs with non-perforated ulcers, survey radiography was usually negative whereas ultrasonography and CT frequently enabled detection of the site of the ulcer; in dogs with perforated ulcers, radiography was frequently positive for peritoneal gas and CT was a sensitive modality for both the ulcer and signs of perforation.

Nitrile biotransformations using free and immobilized cells of a thermophilic Bacillus spp.

A thermophilic Bacillus spp. capable of transforming aliphatic nitriles, cyclic nitriles and dinitriles was used as a free cell suspension and immobilized in alginate beads to study the utilization of acetonitrile and acrylonitrile in a buffered biotransformation medium. The cells grew optimally at 65 degrees C and contained a nitrile hydratase-amidase enzyme system that transformed nitrile compounds stoichiometrically to the corresponding carboxylic acids. In the presence of urea or chloroacetone, amidase activity was inhibited and the amide intermediate was accumulated. Mass transfer limitation of nitrile utilization rates was observed with immobilized cells, but the alginate afforded the cells some degree of additional thermal stability and potential advantage in re-use. In vitro inhibition of the partially purified amidase was confirmed and the use of whole cells of this organism in a continuous bioreactor to generate amide products from nitrile substrates was demonstrated.

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation.

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90-100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO3 and 0.5-2 mg/L BA in combination with 0.01-0.05 mg/L 2,4-D or 0.1-1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All Ro transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R1 seed progeny and showed co-segregation.

Effect of AgNO3 and aminoethoxyvinylglycine on in vitro shoot and root organogenesis from seedling explants of recalcitrant Brassica genotypes.

The presence of 1-10 µM aminoethoxyvinylglycine (AVG) or 5-30 µM AgNO3 markedly enhanced shoot regeneration from cotyledon and hypocotyl cultures of eight recalcitrant Brassica campestris and B. juncea genotypes tested. Expiants of B. campestris ssp. chinensis and ssp. parachinensis grown with a high AVG concentration (20 µM), regenerated poorly. All cytokinins tested were equally effective in promoting shoot formation, except that kinetin was inhibitory to shoot regeneration from hypocotyls of B. campestris ssp. pekinensis (cv. Wong Bok). Both AgNO3 and AVG had no effect on percent rooting and number of roots per rooted cutting of Wong Bok, White Sun and Leaf Heading, but AgNO3 was inhibitory to rooting of India Mustard. However, root elongation of all cuttings was markedly inhibited by AVG at concentrations of 5 and 10 µM.

Structure, organization and expression of transferred DNA in Nicotiana plumbaginifolia crown-gall tissues.

Data are provided which show that transferred DNA (T-DNA) present in Nicotiana plumbaginifolia crown-gall lines in most cases was scrambled and not intact. Both wild-type, and 'rooter'- and 'shooter'-type mutants of octopine-type Agrobacterium tumefaciens were used to infect N. plumbaginifolia plantlets, cultured in vitro. Resulting tumors were excised from the plantlets and cultured for more than three years. During subculturing the tumor lines were scored for the following phenotypic traits: phytohormone autonomous growth in vitro (Aut(+)), spontaneous shoot regeneration (Reg(+)), root deficiency of shoots (Rod(+)), octopine production (Ocs(+)) and mannopine and agropine production (Mas(+)Ags(+)). An unexpectedly large variety of phenotypes was observed. For instance, two out of three tumor lines induced on haploid plantlets by the rooter mutant LBA4210 regenerated shoots, a phenomenon which is not observed for octopine tobacco tumor lines. Fifty percent of the crown-gall lines studied did not contain octopine. Only one line out of six independent lines analyzed was found to have a 'regular' T-DNA structure. Occurrence of aberrant T-DNA structures was not correlated with the ploidy level of infected plantlets, nor with the T-region structure of the inciting bacterial strain. The pattern of TL-DNA transcripts was studied for one line and correlated well with the aberrant T-DNA structure detected. Segments of TR-DNA, having irregular structures as well, were detected in two out of the six lines studied. The scrambled nature of the TR-DNA explained the absence of mannopine and agropine in these two lines. In addition, it was observed that N. plumbaginifolia tissue lines which did not carry T-DNA, became readily phytohormone autotrophic (habituated) at an early stage in tissue culture.

Plant regeneration from protoplasts of long term haploid suspension cultures of N. plumbaginifolia.

A predominantly haploid cell suspension culture of N.plumbaginifolia has been established. The ploidy has remained stable for nearly four years in culture (and is similar in cells recovered after preservation in liquid nitrogen). Protoplasts isolated from these cells regenerate into plants with a high frequency.

Transformation of protoplast-derived cell colonies and suspension cultures by Agrobacterium tumefaciens.

In this paper we describe procedures for transforming micro colonies derived from mesophyll protoplasts of Petunia hybrida with Agrobacterium tumefaciens. The method is efficient, up to 70% of the colonies were transformed, and we used a similar method to transform cells from a suspension culture of haploid Nicotiana plumbaginifolia.

The toxicity of antibiotics to plant cell cultures.

We have examined the toxicity of over twenty antibiotics to protoplast-derived cells of Nicotiana plumbaginifolia. The least toxic antibiotics are the betalactams: ampicillin, carbenicillin and the cephalosporins can be used to provide broad spectrum antimicrobial activity without significant toxicity to plant cells. Similar broad spectrum activity can also be obtained by combining rifampicin and trimethoprim. Other antibiotics which may be useful are erythromycin and colistin. The aminoglycosides are not recommended.