Establishment of in vitro fast-growing normal root culture of Vernonia amygdalina - a potent African medicinal plant

Fast-growing normal root culture of Vernonia amygdalina, a potent African medicinal plant was established from leaf explants of in vitro raised shoot induced from the stem nodal segments on murashige and skoog (MS) medium containing 0.5 mg l-1 6-benzylaminopurine (BA) in combination with 0.5 mg l-1 naphthalene acetic acid (NAA). In vitro raised plantlets were maintained on MS agar medium and sub cultured at 4 weeks interval and used as leaf explant source. Explants were cultured on halfstrengthMS medium supplemented with different concentrations of Indole-3-acetic acid (IAA), indole-3- butyric acid (IBA) and NAA. Basal medium supplemented with IBA at 0.25 and 2.0 mg l-1 and under 16photoperiod condition favoured induction of the longest root (2.7 ± 1.1 cm) and highest number of roots/explant (38.3 ± 1.1) respectively. After 6 weeks well established roots were separated. Fresh root tissue, in amount of a 100 mg were cultured in 50 ml full-strength MS liquid medium supplemented with 2.0 mg l-1 IBA and under continuous agitation (80 rpm). The biomass of root culture was increased to 2.1949 g after 5 weeks of culture. The root culture was maintained up to 6 weeks. The protocoldeveloped in this study provides a basis for adventitious root induction and for further investigation of medicinally active constituents of this elite medicinal plant

Efficient production of transgenic soybean (Glycine max [L] Merrill) plants mediated via whisker-supersonic (WSS) method

The present study was designed to evaluate the transformation efficiency and proof the capability of whisker supersonic (WSS) method as an alternative option for soybean (Glycine max [L] Merrill)transformation. We compared soybean transformation efficiency obtained by WSS-mediated with that of particle bombardment transformation by carrying out molecular analysis of the T0 plants in two independent experiments. For this, we used for both transformation techniques the same genotype, the same plasmid and the same selection method. To assess the efficiency of soybean genetictransformation, we evaluated the efficiency of multi gene transformation by the selection with hygromycin and the expression of green fluorescent protein [sGFP (S65T)] resulted from both techniques. Regenerable embryogenic cells were induced from immature cotyledons of soybean c.v Jack on MSD40 media within 3 weeks then proliferated on FN lite liquid media and engineered with pUHG gene construct through both WSS and particle bombardment-mediated transformation. The pUHG was constructed with pUC 19 and contain the hpt gene conferring resistance to hygromycin as a selective marker and sGFP(S65T) as a reporter gene. Fluorescence microscopy screening after the selection of hygromycin, identified the clearly expression of sGFP(S65T) in the transformed soybean embryos. Stable integration of the transgenes was confirmed by polymerase chain reaction (PCR) andSouthern blot analysis. The average transformation efficiency achieved with WSS was higher than that obtained by particle bombardment and hence it may represent an alternative method for soybeantransformation

Willow Leaves' Extracts Contain Anti-Tumor Agents Effective against Three Cell Types

Many higher plants contain novel metabolites with antimicrobial, antifungal and antiviral properties. However, in the developed world almost all clinically used chemotherapeutics have been produced by in vitro chemical synthesis. Exceptions, like taxol and vincristine, were structurally complex metabolites that were difficult to synthesize in vitro. Many non-natural, synthetic drugs cause severe side effects that were not acceptable except as treatments of last resort for terminal diseases such as cancer. The metabolites discovered in medicinal plants may avoid the side effect of synthetic drugs, because they must accumulate within living cells. The aim here was to test an aqueous extract from the young developing leaves of willow (Salix safsaf, Salicaceae) trees for activity against human carcinoma cells in vivo and in vitro. In vivo Ehrlich Ascites Carcinoma Cells (EACC) were injected into the intraperitoneal cavity of mice. The willow extract was fed via stomach tube. The (EACC) derived tumor growth was reduced by the willow extract and death was delayed (for 35 days). In vitro the willow extract could kill the majority (75%–80%) of abnormal cells among primary cells harvested from seven patients with acute lymphoblastic leukemia (ALL) and 13 with AML (acute myeloid leukemia). DNA fragmentation patterns within treated cells inferred targeted cell death by apoptosis had occurred. The metabolites within the willow extract may act as tumor inhibitors that promote apoptosis, cause DNA damage, and affect cell membranes and/or denature proteins

Diffusion of Protease into Meat & Bone Meal for Solubility Improvement and Potential Inactivation of the BSE Prion

BACKGROUND: Government-imposed feed bans have created a need for new applications for meat & bone meal (MBM). Many potential new applications require MBM protein to be both soluble and free of infectious prion. Treatment with protease is generally effective in reducing insoluble, thermally-denatured proteins to soluble peptides. It has been reported in the literature that certain proteases, including Versazyme™, are able to degrade infectious prions in a system where the prions are readily accessible to proteolytic attack. Prions distributed within MBM, however, may conceivably be protected from proteases. METHODOLOGY/PRINCIPAL FINDINGS: The overall rate of proteolytic MBM digestion depends greatly on whether the protease can penetrate deep within individual particles, or if the protease can only act near the surface of the particle. This research examined the barriers to the diffusion of Versazyme™ into particles of MBM. Confocal microscopy demonstrated differences in the density distributions between the bone and the soft tissue particles of MBM. By tracking the diffusion of fluorescently labeled Versazyme™ through individual particles, it was found that bone particles show full Versazyme™ penetration within 30 minutes, while penetration of soft tissue particles can take up to four hours, depending on the particle's diameter. From the variety of normal proteins comprising MBM, a specific protein was chosen to serve as a prion surrogate based on characteristics including size, solubility, distribution and abundance. This surrogate was used to measure the effect of several factors on Versazyme™ diffusion. CONCLUSIONS/SIGNIFICANCE: Results showed that surrogate distributed in bone particles was more susceptible to degradation than that in soft tissue particles. Three factors controllable by unit operations in an industrial-scale process were also tested. It was found that removing the lipid content and hydrating MBM prior to incubation both significantly increased the rate of surrogate degradation. In a test of particle size, the smallest collected diameter range demonstrated the largest degradation of the prion surrogate, suggesting milling would be beneficial

Computer-Aided Lead Optimization: Improved Small-Molecule Inhibitor of the Zinc Endopeptidase of Botulinum Neurotoxin Serotype A

Optimization of a serotype-selective, small-molecule inhibitor of botulinum neurotoxin serotype A (BoNTA) endopeptidase is a formidable challenge because the enzyme-substrate interface is unusually large and the endopeptidase itself is a large, zinc-binding protein with a complex fold that is difficult to simulate computationally. We conducted multiple molecular dynamics simulations of the endopeptidase in complex with a previously described inhibitor (Kiapp of 7±2.4 µM) using the cationic dummy atom approach. Based on our computational results, we hypothesized that introducing a hydroxyl group to the inhibitor could improve its potency. Synthesis and testing of the hydroxyl-containing analog as a BoNTA endopeptidase inhibitor showed a twofold improvement in inhibitory potency (Kiapp of 3.8±0.8 µM) with a relatively small increase in molecular weight (16 Da). The results offer an improved template for further optimization of BoNTA endopeptidase inhibitors and demonstrate the effectiveness of the cationic dummy atom approach in the design and optimization of zinc protease inhibitors

Models Analyses for Allelopathic Effects of Chicory at Equivalent Coupling of Nitrogen Supply and pH Level on F. arundinacea, T. repens and M. sativa

Alllelopathic potential of chicory was investigated by evaluating its effect on seed germination, soluble sugar, malondialdehyde (MDA) and the chlorophyll content of three target plants species (Festuca arundinacea, Trifolium repens and Medicago sativa). The secretion of allelochemicals was regulated by keeping the donor plant (chicory) separate from the three target plant species and using different pH and nitrogen levels. Leachates from donor pots with different pH levels and nitrogen concentrations continuously irrigated the target pots containing the seedlings. The allelopathic effects of the chicory at equivalent coupling of nitrogen supply and pH level on the three target plants species were explored via models analyses. The results suggested a positive effect of nitrogen supply and pH level on allelochemical secretion from chicory plants. The nitrogen supply and pH level were located at a rectangular area defined by 149 to 168 mg/l nitrogen supply combining 4.95 to 7.0 pH value and point located at nitrogen supply 177 mg/l, pH 6.33 when they were in equivalent coupling effects; whereas the inhibitory effects of equivalent coupling nitrogen supply and pH level were located at rectangular area defined by 125 to 131 mg/l nitrogen supply combining 6.71 to 6.88 pH value and two points respectively located at nitrogen supply 180 mg/l with pH 6.38 and nitrogen supply 166 mg/l with pH 7.59. Aqueous extracts of chicory fleshy roots and leaves accompanied by treatment at different sand pH values and nitrogen concentrations influenced germination, seedling growth, soluble sugar, MDA and chlorophyll of F. arundinacea, T. repens and M. sativa. Additionally, we determined the phenolics contents of root and leaf aqueous extracts, which were 0.104% and 0.044% on average, respectively

Cattle Mammary Bioreactor Generated by a Novel Procedure of Transgenic Cloning for Large-Scale Production of Functional Human Lactoferrin

Large-scale production of biopharmaceuticals by current bioreactor techniques is limited by low transgenic efficiency and low expression of foreign proteins. In general, a bacterial artificial chromosome (BAC) harboring most regulatory elements is capable of overcoming the limitations, but transferring BAC into donor cells is difficult. We describe here the use of cattle mammary bioreactor to produce functional recombinant human lactoferrin (rhLF) by a novel procedure of transgenic cloning, which employs microinjection to generate transgenic somatic cells as donor cells. Bovine fibroblast cells were co-microinjected for the first time with a 150-kb BAC carrying the human lactoferrin gene and a marker gene. The resulting transfection efficiency of up to 15.79×10−2 percent was notably higher than that of electroporation and lipofection. Following somatic cell nuclear transfer, we obtained two transgenic cows that secreted rhLF at high levels, 2.5 g/l and 3.4 g/l, respectively. The rhLF had a similar pattern of glycosylation and proteolytic susceptibility as the natural human counterpart. Biochemical analysis revealed that the iron-binding and releasing properties of rhLF were identical to that of native hLF. Importantly, an antibacterial experiment further demonstrated that rhLF was functional. Our results indicate that co-microinjection with a BAC and a marker gene into donor cells for somatic cell cloning indeed improves transgenic efficiency. Moreover, the cattle mammary bioreactors generated with this novel procedure produce functional rhLF on an industrial scale

New Insights into the Organization, Recombination, Expression and Functional Mechanism of Low Molecular Weight Glutenin Subunit Genes in Bread Wheat

The bread-making quality of wheat is strongly influenced by multiple low molecular weight glutenin subunit (LMW-GS) proteins expressed in the seeds. However, the organization, recombination and expression of LMW-GS genes and their functional mechanism in bread-making are not well understood. Here we report a systematic molecular analysis of LMW-GS genes located at the orthologous Glu-3 loci (Glu-A3, B3 and D3) of bread wheat using complementary approaches (genome wide characterization of gene members, expression profiling, proteomic analysis). Fourteen unique LMW-GS genes were identified for Xiaoyan 54 (with superior bread-making quality). Molecular mapping and recombination analyses revealed that the three Glu-3 loci of Xiaoyan 54 harbored dissimilar numbers of LMW-GS genes and covered different genetic distances. The number of expressed LMW-GS in the seeds was higher in Xiaoyan 54 than in Jing 411 (with relatively poor bread-making quality). This correlated with the finding of higher numbers of active LMW-GS genes at the A3 and D3 loci in Xiaoyan 54. Association analysis using recombinant inbred lines suggested that positive interactions, conferred by genetic combinations of the Glu-3 locus alleles with more numerous active LMW-GS genes, were generally important for the recombinant progenies to attain high Zeleny sedimentation value (ZSV), an important indicator of bread-making quality. A higher number of active LMW-GS genes tended to lead to a more elevated ZSV, although this tendency was influenced by genetic background. This work provides substantial new insights into the genomic organization and expression of LMW-GS genes, and molecular genetic evidence suggesting that these genes contribute quantitatively to bread-making quality in hexaploid wheat. Our analysis also indicates that selection for high numbers of active LMW-GS genes can be used for improvement of bread-making quality in wheat breeding

Aloe barbadensis: how a miraculous plant becomes reality

Aloe barbadensis Miller is a plant that is native to North and East Africa and has accompanied man for over 5,000 years. The aloe vera plant has been endowed with digestive, dermatological, culinary and cosmetic virtues. On this basis, aloe provides a range of possibilities for fascinating studies from several points of view, including the analysis of chemical composition, the biochemistry involved in various activities and its application in pharmacology, as well as from horticultural and economic standpoints. The use of aloe vera as a medicinal plant is mentioned in numerous ancient texts such as the Bible. This multitude of medicinal uses has been described and discussed for centuries, thus transforming this miracle plant into reality. A summary of the historical uses, chemical composition and biological activities of this species is presented in this review. The latest clinical studies involved in vivo and in vitro assays conducted with aloe vera gel or its metabolites and the results of these studies are reviewed