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     

<Emphasis Type="Italic">Lupinus albus</Emphasis> Conglutin Gamma Modifies the Gene Expressions of Enzymes Involved in Glucose Hepatic Production <Emphasis Type="Italic">In Vivo</Emphasis>

Lupinus albus seeds contain conglutin gamma (Cγ) protein, which exerts a hypoglycemic effect and positively modifies proteins involved in glucose homeostasis. Cγ could potentially be used to manage patients with impaired glucose metabolism, but there remains a need to evaluate its effects on hepatic glucose production. The present study aimed to analyze G6pc, Fbp1, and Pck1 gene expressions in two experimental animal models of impaired glucose metabolism. We also evaluated hepatic and renal tissue integrity following Cγ treatment. To generate an insulin resistance model, male Wistar rats were provided 30% sucrose solution ad libitum for 20 weeks. To generate a type 2 diabetes model (STZ), five-day-old rats were intraperitoneally injected with streptozotocin (150 mg/kg). Each animal model was randomized into three subgroups that received the following oral treatments daily for one week: 0.9% w/v NaCl (vehicle; IR-Ctrl and STZ-Ctrl); metformin 300 mg/kg (IR-Met and STZ-Met); and Cγ 150 mg/kg (IR-Cγ and STZ-Cγ). Biochemical parameters were assessed pre- and post-treatment using colorimetric or enzymatic methods. We also performed histological analysis of hepatic and renal tissue. G6pc, Fbp1, and Pck1 gene expressions were quantified using real-time PCR. No histological changes were observed in any group. Post-treatment G6pc gene expression was decreased in the IR-Cγ and STZ-Cγ groups. Post-treatment Fbp1 and Pck1 gene expressions were reduced in the IR-Cγ group but increased in STZ-Cγ animals. Overall, these findings suggest that Cγ is involved in reducing hepatic glucose production, mainly through G6pc inhibition in impaired glucose metabolism disorders.     

Proteomic profiles during adventitious rooting of Eucalyptus species relevant to the cellulose industry

New Forests - Adventitious rooting (AR) is an obligatory step for vegetative propagation of commercial woody species. Paper industries have interest in Eucalyptus globulus Labill and its hybrids...     

Evaluation of Particle Size Influence on Proximate Composition,Physicochemical, Techno-Functional and Physio-Functional Properties of Flours Obtained from Persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Trumb.) Coproducts

The aim of the work was to study the influence of particle size in the composition, physicochemical, techno-functional and physio-functional properties of two flours obtained from persimmon (Diospyros kaki Trumb. cvs. ‘Rojo Brillante’ (RBF) and ‘Triump’ (THF) coproducts. The cultivar (RBF and THF) and particle size significantly affected all parameters under study, although depending on the evaluated property, only one of these effects predominated. Carbohydrates (38.07–46.98 g/100 g) and total dietary fiber (32.07–43.57 g/100 g) were the main components in both flours (RBF and THF). Furthermore, insoluble dietary fiber represented more than 68% of total dietary fiber content. All color properties studied were influenced by cultivar and particle size. For both cultivars, the lower particle size, the higher lightness and hue values. RBF flours showed high values for emulsifying activity (69.33–74.00 mL/mL), while THF presented high values for water holding capacity (WHC: 9.47–12.19 g water/g sample). The bile holding capacity (BHC) and fat/oil binding values were, in general, higher in RBF (19.61–12.19 g bile/g sample and 11.98–9.07, respectively) than THF (16.12–12.40 g bile/g sample and 9.78–7.96, respectively). The effect of particle size was really evident in both WHC and BHC. Due to their dietary fiber content, techno-functional and physio-functional properties, persimmon flours seem to have a good profile to be used as potential functional ingredient.     

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.     

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.     

Particle Size Distribution in Two Lipid Emulsions Used for the Enrichment of Artemia nauplii as a Function of Their Preparation Method and Storage Time

The present study examined the distribution of particle sizes in two experimental standardized lipid emulsions (distributed by ICES, International Council for the Exploration of the Sea) as a function of the preparation method (hand shaking or ultrasonic blending) and as a function of storage time. A 24-h enrichment trial compared the incorporated HUFA levels in the nauplii of Artemia franciscana .
The emulsion droplets in the 50% HUFA emulsion (ICES 50, ethyl ester based) were much smaller than in the 30% HUFA emulsion (ICES 30, triacylglycerol-based) in which 90% of the droplets had a diameter below 12.3 μm as compared to 1.3 pn in ICES 50. The blending method highly affected particle sizes. High-shear blending instead of the classical hand shaking of the emulsion of both ICES 30 and 50 reduced the mean particle size from 5.06 to 1.07 μm and from 0.38 to 0.11 μm, respectively. The particle size distributions remained stable during the 1-wk storage, suggesting the absence of agglomeration or bacterial development. The fatty acid composition of 24-h enriched Artemia reflected differences in the HUFA profile of the emulsions, but was independent of observed differences in the size of the particles.     

Blastocyst Evaluation by Means of Differential Staining: a Practical Approach

Techniques for in vitro production of embryos have been developed world-wide in different species, with promising results in human and ruminants. Thousands of human IVF-babies have been born during the last 20 years and thousands of in vitro -produced calves have been born since the late 1980s. With current methods for bovine in vitro fertilization, about 30–40% of in vitro -fertilized bovine oocytes develop further to the blastocyst stage and can be used for transfer. A proper evaluation of blastocyst quality remains however, an important challenge for every researcher involved in embryology and for every clinician who wants to select the best embryos for transfer. This review attempts to summarize the different methods available for estimation of blastocyst quality with a special emphasis upon differential staining.