Agrobacterium-mediated transformation of the CrDAT gene and selection of transgenic periwinkle lines with a high vincristine accumulation

Catharanthus roseus contains vincristine and vinblastine, which are outstanding drugs for cancer. In the biosynthetic pathways of terpenoid indole alkaloids (TIAs) in C. roseus, deacetylvindoline 4-O-acetyltransferase (DAT) is a key enzyme that catalyses the last reaction of vindoline biosynthesis to form vinblastine and vincristine. In this study, the CrDAT transgene was transferred into the periwinkle by Agrobacterium-mediated transformation and generated transgenic periwinkle lines with an increase in vincristine accumulation. The C. roseus DAT gene was introduced into C. roseus plants and it was confirmed that CrDAT was successfully transferred into the genome of periwinkle plants and efficiently translated to synthesise recombinant DAT protein. Four transgenic periwinkle lines in T1 generation, T1-1, T1-3, T1-6, and T1-7, expressed recombinant DAT protein with the total protein content in the range of 2.86 μg.mg^?1 to 5.12 μg.mg^?1. Moreover, the vincristine contents of four transgenic lines increased by 1.63?2.48-fold compared to non-transgenic plants, ranging from 6.91 µg.g^?1 (fresh weight) to 10.53 µg.g^?1 (fresh weight). The T1-1 line had the highest vincristine content. Hence, the overexpression of the recombinant DAT protein can improve the vincristine accumulation of transgenic C. roseus plants.

Abbreviation: CrDAT - Catharanthus roseus Deacetylvindoline-4-O-Acetyl Transferase; D4H - Deacetoxyvindoline 4-hydroxylase; ELISA - Enzyme-Linked Immunosorbent Assay Monoterpene indole alkaloid; T0, T1 - Generations of transgenic plants; TIAs - Terpenoid indole alkaloids; WT- The wild-type tobacco plants (non transgenic plant); 35S - Cauliflower mosaic virus 35S promoter     

No effect of turkey herpesvirus infections on the outcome of avian pneumovirus infections in turkeys.

To determine whether turkey herpesvirus (HVT) impairs the aspecific and specific defense against an avian pneumovirus (APV) infection, specific-pathogen-free turkeys were inoculated at 7 days of age with HVT and 1, 5, or 7 wk later with APV. Clinical signs, APV replication, and development of antibodies against APV were evaluated. No differences were found between the birds that received both HVT and APV and those that received only APV.     

Culture of bovine bone marrow progenitor cells in vitro.

In vitro methylcellulose cultures of bovine bone marrow progenitor cells were developed. An existing technique described for bovine species was compared to a method for human tissue and further adapted during subsequent experiments. Bovine bone marrow samples were collected at the slaughterhouse, and mononuclear cells were separated by gradient centrifugation (1.077 g/ml specific density and 400 g). The use of 3% bovine leucocyte-conditioned medium, produced by stimulation of blood lymphocytes with 4 microg/ml concanavalin A and harvested on day 4 of culture, gave better results than the use of supernatant of the human bladder carcinoma 5637, which is widely used in human bone marrow cultures. However, bovine leucocyte-conditioned medium was not added to erythroid cultures because inhibitory effects were observed. Erythroid colonies were stimulated with erythropoietin, and hemin was added to enable microscopic identification. Reduced oxygen tension was necessary to induce growth of erythroid colonies. This was not necessary for myeloid cultures. In conclusion, the results of this study show that the growth of myeloid and erythroid colonies in methylcellulose-based medium requires different culture conditions, which are different from the culture conditions for human cells.     

Evaluation of an enzyme-linked immunosorbent assay for diagnosis of trichinellosis in swine.

Experimental and field trials were conducted to evaluate an ELISA for its ability to detect Trichinella-infected domestic swine and to compare ELISA results with muscle-digestion test results. The ELISA used was a commercial double-antibody kit, containing an excretory-secretory antigen, and was evaluated principally for epidemiologic use. Experimentally induced infection in swine (4 groups of 3 pigs each; inoculated with 0, 50, 500 or 5,000 larvae) was detected as early as postinoculation week 4, with seroconversion of all inoculated swine by postinoculation week 8. The rate of seroconversion appeared to be affected by initial larval dose, time after inoculation, and immunocompetence of the individual host. Determination of antibody kinetics generally revealed rapidly increasing antibody titer, followed by its steady decrease in most pigs. Once seropositive, however, all pigs remained seropositive for the duration of the 10-week study. Presence of muscle larvae was confirmed in all infected pigs at termination of the study. We recognize that the experimental conditions may not be truly representative of those under which natural infection develops in pigs; however, the ELISA detected an infected pig with muscle larval density of 0.87 larvae/g of tissue. Results of a field trial (n = 310) indicated no muscle digestion test-positive pigs (35 g of diaphragm muscle digested/pig), but 3 samples tested positive by ELISA for specificity of 99.0%.     

A Tremor Syndrome With a Central Axonopathy in Scottish Terriers

A central axonopathy in 2 male and 1 female Scottish Terrier puppies from 3 different but related litters is reported. Clinical signs consisting of severe whole-body tremors and ataxia were first detected at the age of 10 to 12 weeks. They worsened with activity and excitement and diminished during rest or sleep. Two dogs also had paraparesis. In 1 dog the neurological deficits progressed over several months. Neuropathological examination revealed widespread axonal changes, vacuolation, and gliosis in the white matter of the central nervous system.     

Hyperadrenocorticism in four cats.

This paper describes four cats with hyperadrenocorticism. Cat 1 showed polydipsia and polyphagia. Diabetes mellitus was initially diagnosed. As the animal appeared to be insulin resistant, pituitary and adrenocortical function tests were performed and the diagnosis of hyperadrenocorticism was made. Resistance to the high-dose dexamethasone suppression test was noticed in this cat. Pathological examination revealed a pituitary chromophobe adenoma. Cat 2 presented with diabetes mellitus, which was treated with insulin. The animal had a pendulous abdomen and its coat was in a poor condition. The low-dose dexamethasone suppression test demonstrated hyperadrenocorticism. Necropsy findings of pituitary tumour and hyperplasia of the adrenal cortex confirmed the diagnosis. Cat 3 showed clinical abnormalities indicative of hyperadrenocorticism, for instance, muscle weakness, alopecia, multiple abscesses. The diagnosis of hyperadrenocorticism was confirmed by the results of the lowe-dose dexamethasone suppression test. Pathological examination revealed an adrenocortical carcinoma. Cat 4 presented with polydipsia. The cause of this symptom was not found initially. One and a half years later additional symptoms, such as nephritis and polyphagia developed. Hyperadrenocorticism was diagnosed because of a palpable mass cranial to the left kidney. The diagnosis was confirmed by the results of the lowe-dose dexamethasone suppression test and the necropsy findings.     

Comparison of sesame cake and cottonseed as supplementary sources of protein to weaned N'Dama bull calves in The Gambia

Summary The potential for increased productivity of N'Dama cattle in response to small supplementary inputs of proteinaceous by-products has been shown to be substantial under village husbandry conditions in The Gambia. Young N'Dama bulls were used to compare sesame cake with cottonseed as sources of supplementary protein during the wet and early dry seasons of 1987. They were fed 0, 40, 80 or 160 g crude protein/day in these forms for four months. Supplementation significantly increased growth rate from 169 g/d in controls to means of 272 and 271 g/d for those given sesame cake and cottonseed respectively; there were no significant differences among the supplemented groups. During the four months of the dry season immediately after supplementation ceased all previously supplemented groups grew significantly more rapidly than the controls (at leastP<0·05), indicating a substantial carry over effect of the supplements. Over this period the mean gain of the groups previously given sesame cake exceeded that of those that had received cottonseed (217 cf. 158 g/d;P<0·01). The economic benefits of these responses were substantial.

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Comparacion de torta de sesamo y de semilla de algodon como fuente suplementaria de proteina para terneros destetos N'Dama en Gambia

Resumen Se ha demonstrado el potencial para incrementar la productividad del ganado N'Dama, como resquesta a peque?as cantidades de insumos suplementarios de proteína, bajo condiciones de villorrio en Gambia. Se utilizaron terneros destetos N'Dama par comparar la torta de sésamo y de semilla de algodón como fuente de proteína suplementaria durate la estaicón lluviosa y principio de la seca del a?o 1987. Se les dió a los animales 0, 40, 80 o 160 g de proteína cruda/día en éstas formas durante cuatro meses. La suplementación incrementó significativamente la tasa de crecimiento, de 169 g/día en controles a 272 y 271 g/día en aquellos que recibieron torta de sésamo y de algodón respectivamente; no hubo diferenca estadística entre los grupos suplementados. Durate los cuatro meses de la estación seca inmediatamente después de que la suplementación fue suspendida, todos los grupos que habían sido suplementados crecieron sgnificativamente más rápido que los controls (P<0·05), indicando un efecto compensatorio en los suplementados. Durante ese período la media de crecimento de los grupos que recibieron torta de sésamo excedió a aquella de los que habían recibido torta de algodón (217 cf. 158 g/d;P<0·01). Los beneficios económicos de estos resultados fueron substanciales.

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Comparaison entre le tourteau de sesame et la graine de coton comme sources complementaires de proteines pour les taurillons N'Dama sevres en Gambie

Résumé Le potentiel de réponse à de petites quantités supplémentaires de sous-produits proteiniques pour accroitre la productivité de bétail N'Dama, s'est révélé manifeste, dans les conditions d'élevage villageois en Gambie. De jeunes taureaux N'Dama ont été utilisés pour comparer le tourteau de sésame et la graine de coton en tant que source de complémentation protéinique pendant la saison sèche et le début de la saison humide au cours de l'année 1987. Ils ont re?u respectivement 0, 40, 80 ou 160 g de proteines brutes par jour sus ces deux formes pendat quatre mois. Cette supplémentation a accru de fa?on significative le taux de croissance, de 169 g/jour pour les témoins jusqu'à des moyennes de 272 et 271g/jour respectivement pour ceux qui ont re?u du tourteau de sésame et de la graine de coton. Quant aux groupes complémentés, il n'ont révélé aucune différence significative. Pendat les quatre mois de la saison sèche et immédiatement après l'arrêt de la supplémentation, tous les lots ayant fait précédemment l'objet d'une distribution complémentaire ont eu une croissance significativement plus rapide que les animaux témoins, du moins pourP<0,05, ce qui indique un effet important de report des suppléments. Durant cette période, le gain moyen des lots qui ont re?u le tourteau de sésame a dépassé celui des lots auxquels on a donné de la graine de coton, soit 217 g/jour contre 158 g/jour pourP<0,01. Les profits économiques de ces réponses ont été appréciables.

     

Research note: Postnatal development of electrolyte transport in calf rumen as affected by weaning time.

In a previous study we found a positive correlation between early weaning in calves and morphological parameters which were indicative of ruminal development, i.e. the length and width of the papillae. The objective of the present study was to determine to what extent this observation could be reflected by modulations of absorptive and secretory functions of the rumen mucosa. For this purpose the short-circuit currents (Isc) as a measure of electrogenic net ion fluxes and the transepithelial conductances (G(T)) as a measure of the overall tissue permeability were measured in vitro applying the Ussing-chamber technique. Simultaneously, the unidirectional flux rates of sodium and chloride across rumen wall epithelia were determined in the absence of electrochemical gradients. Under these conditions, significant positive net flux rates (Jnet) clearly indicate active mechanisms for electrolyte absorption. For the experiments 12 male Holstein calves 7 d of age were assigned to three groups of 4 animals each: milk group (I, slaughtered after 6 weeks of age), late weaning group (II, slaughtered after 9 weeks of age) and early weaning group (II, weaned after 6 weeks of age and slaughtered after 9 weeks of age). Whereas G(T) values remained unaffected by different age and feeding, Isc values were significantly affected by early weaning but were not influenced by age. Irrespective of weaning time active absorption of Na+ tended to be higher by about 60% in 9 weeks old animals. Active absorption of chloride was significantly increased in milk fed 9 weeke old calves and this effect was further stimulated by early weaning. In conclusion, the data show an increasing active Na+ absorption with age in calf rumen that could not be influenced by early weaning. Similarly, active Cl- absorption was initially increased during postnatal development and this effect could be stimulated further by early weaning.