Ratiometric bioassay and visualization of dopamine ß-hydroxylase in brain cells utilizing a nanohybrid fluorescence probe.

Dopamine ß-hydroxylase (DBH) is involved in various neuronal transmission processes in the brain. Due to the severe diseases caused by abnormity levels of such important enzyme in human serum, sensitive and rapid detection of DBH at early stages is crucial, particularly for clinical analysis. Herein, we developed optical sensors for DBH that include the following: (i) a ratiometric fluorescence sensor that hybridizes the bovine serum albumin (BSA)-gold nanoclusters (BSA-AuNCs) and nitrogen doped carbon dots (N-CDs). The sensor proved to be highly selective and sensitive, achieving a linear range of 0.02-0.16 µg mL-1 and a limit of detection of 4.0 ng mL-1. In the presence of DBH, the fluorescence intensity of BSA-AuNCs (λem = 615 nm) was remarkably quenched by DBH serving as a reporter signal, whereas the N-CDs fluorescence intensity at 440 nm was almost kept unchanged serving as a reference signal. The developed ratiometric sensor is capable of demonstrating a color change from pink to violet and blue with a gradual increase in DBH concentration, which is discernible by the naked-eye. A test strip is prepared for semi-quantitative assay and convenient use. Intriguingly, by taking advantage of the inter-AuNCs aggregation in the presence of DBH, (ii) a resonance light scattering (RLS) sensor was also developed based on the nanohybrid probe (detection limit 95 ng mL-1). Fluorescence imaging in PC12 cell lines demonstrated that the BSA-AuNCs could be utilized in visualization assay towards intracellular DBH. Additionally, the sensors were tested in a real matrix by spiking serum samples with satisfactory recoveries.

Expression of active chimeric-tissue plasminogen activator in tobacco hairy roots, identification of a DNA aptamer and purification by aptamer functionalized-MWCNTs chromatography.

Tissue-type plasminogen activator (tPA) is a serine protease that plays a crucial role in the fibrinolytic system. We increased the activity of tPA by splicing the active site of dodder-cuscutain gene to human tPA. The chimeric cDNA of tPA was constructed by Splicing by Overlap Extension Polymerase Chain Reaction (SOEing-PCR) method and transferred to the hairy roots of tobacco using different strains of Agrobacterium rhizogenes. Chimeric-tPA was purified by lysine-sepharose chromatography and specific aptamers were designed using SELEX method. Multi wall carbon nanotubes were functionalized with selected aptamers, packed in a column, and used for purification. The results demonstrated that selected aptamer having KD values of 0.320 nM and IC50 of 28.9 nM possessed good affinity to tPA, and the chimeric-tPA was properly purified by aptamer-chromatography. Hairy roots expressing chimeric-tPA and normal-tPA produced 900 and 450 ngmg-1 of total protein, respectively. The activities of chimeric-tPA and normal-tPA were 90 and 60 IUml-1, respectively. Compared to the normal-tPA, chimeric-tPA showed more activity.

Improvement of in vitro embryo maturation, plantlet regeneration and transformation efficiency from alfalfa (Medicago sativa L.) somatic embryos using Cuscuta campestris extract.

Developmental deficiency of somatic embryos and regeneration to plantlets, especially in the case of transformation, are major problems of somatic embryo regeneration in alfalfa. One of the ways to overcome these problems is the use of natural plant regulators and nutrients in the culture medium of somatic embryos. For investigating the influence of Cuscuta campestris extract on the efficiency of plant regeneration and transformation, chimeric tissue type plasminogen activator was transferred to explants using Agrobacterium tumefaciens, and transgenic plants were recovered using medium supplemented with different concentration of the extract. Transgenic plants were analyzed by PCR and RT-PCR. Somatic embryos of Medicago sativa L. developed into plantlets at high frequency level (52 %) in the maturation medium supplemented with 50 mg 1-1C. campestris extract as compared to the medium without extract (26 %). Transformation efficiency was 29.3 and 15.2 % for medium supplemented with dodder extract and without the extract, respectively. HPLC and GC/MS analysis of the extract indicated high level of ABA and some compounds such as Phytol, which can affect the somatic embryo maturation. The antibacterial assay showed that the extract was effective against some strains of A. tumefaciens. These results have provided a scientific basis for using of C. campestris extract as a good natural source of antimicrobial agents and plant growth regulator as well, that can be used in tissue culture of transgenic plants.

Expression analysis and purification of human recombinant tissue type plasminogen activator (rt-PA) from transgenic tobacco plants.

Recombinant tissue-type plasminogen activator (rt-PA) has been produced in different hosts. In this research, transgenic tobacco was selected for production of human rt-PA. Transgenic plants were analyzed by polymerase chain reaction (PCR) and reverse-transcription (RT)-PCR. The protein was extracted by Lysine Sepharose chromatography column and was further purified by HiTrap desalting column. The function of eluted protein was analyzed on zymography gel. The results showed that the 1.7-kb cDNA of tissue-type plasminogen activator (t-PA) (as well as a shortened 650-bp transcript of t-PA) has been expressed in transgenic plants. The anticipated 63-kD protein band and an additional 53-kD protein were observed in transgenic plants. Finally, zymography assay revealed that the purified rt-PA has anticipated appropriate activity comparable to a positive control drug (Alteplase). On the whole, we can say that transgenic tobacco is a good alternative host for production of t-PA.