An Untapped Resource in the Spotlight of Medicinal Biotechnology: The Genus Scutellaria.

BACKGROUND: This review is intended to draw the attention of pharmaceutical and biotechnological communities to the untapped potential of the Scutellaria genus. Skullcaps, as they are more widely known, are found in one of the oldest materia medica in the world, that of ancient Chinese pharmacology, and their numerous wide range of medicinal bioactivities have been studied both in vivo and in vitro. For thousands of years, chemical compounds from the Scutellaria species have been safely used as antitumor, antibacterial, antiviral, anti-inflammatory, antioxidant or hepatoprotective factors. OBJECTIVE: As these effects are well known, reflected in the presence of Scutellaria plants in national pharmacopoeias, it is clear that the plant has yet enormous unexploited potential. The European pharmacological market has turned to the resources of Scutellaria only in the last two decades, and although the construction and clinical processing of a new drug is a long process, the general impression is that very few medical products in pharmacies have been inspired by the phytochemistry of skullcaps. CONCLUSION: This paper presents the current state of knowledge on the wealth of Scutellaria chemical compounds with treatment applications, its tissue culture and biotechnological achievements, especially in the context of the production of secondary metabolites.

The pharmaceutics from the foreign empire: the molecular pharming of the prokaryotic staphylokinase in Arabidopsis thaliana plants.

Here, we present the application of microbiology and biotechnology for the production of recombinant pharmaceutical proteins in plant cells. To the best of our knowledge and belief it is one of few examples of the expression of the prokaryotic staphylokinase (SAK) in the eukaryotic system. Despite the tremendous progress made in the plant biotechnology, most of the heterologous proteins still accumulate to low concentrations in plant tissues. Therefore, the composition of expression cassettes to assure economically feasible level of protein production in plants remains crucial. The aim of our research was obtaining a high concentration of the bacterial anticoagulant factor-staphylokinase, in Arabidopsis thaliana seeds. The coding sequence of staphylokinase was placed under control of the ß-phaseolin promoter and cloned between the signal sequence of the seed storage protein 2S2 and the carboxy-terminal KDEL signal sequence. The engineered binary vector pATAG-sak was introduced into Arabidopsis thaliana plants via Agrobacterium tumefaciens-mediated transformation. Analysis of the subsequent generations of Arabidopsis seeds revealed both presence of the sak and nptII transgenes, and the SAK protein. Moreover, a plasminogen activator activity of staphylokinase was observed in the protein extracts from seeds, while such a reaction was not observed in the leaf extracts showing seed-specific activity of the ß-phaseolin promoter.

Phaseolus vulgaris - recalcitrant potential.

Since the ability to genetically engineer plants was established, researchers have modified a great number of plant species to satisfy agricultural, horticultural, industrial, medicinal or veterinary requirements. Almost thirty years after the first approaches to the genetic modification of pulse crops, it is possible to transform many grain legumes. However, one of the most important species for human nutrition, Phaseolus vulgaris, still lacks some practical tools for genomic research, such as routine genetic transformation. Its recalcitrance towards in vitro regeneration and rooting significantly hampers the possibilities of improvement of the common bean that suffers from many biotic and abiotic constraints. Thus, an efficient and reproducible system for regeneration of a whole plant is desired. Although noticeable progress has been made, the rate of recovery of transgenic lines is still low. Here, the current status of tissue culture and recent progress in transformation methodology are presented. Some major challenges and obstacles are discussed and some examples of their solutions are presented.

Expression of recombinant staphylokinase, a fibrin-specific plasminogen activator of bacterial origin, in potato (Solanum tuberosum L.) plants.

One of the most dynamically developing sectors of green biotechnology is molecular farming using transgenic plants as natural bioreactors for the large scale production of recombinant proteins with biopharmaceutical and therapeutic values. Such properties are characteristic of certain proteins of bacterial origin, including staphylokinase. For many years, work has been carried out on the use of this protein in thrombolytic therapy. In this study, transgenic Solanum tuberosum plants expressing a CaMV::sak-mgpf-gusA gene fusion, were obtained. AGL1 A. tumefaciens strain was used in the process of transformation. The presence of the staphylokinase gene was confirmed by PCR in 22.5% of the investigated plants. The expression of the fusion transgene was detected using the ß-glucuronidase activity assay in 32 putative transgenic plants. Furthermore, on the basis of the GUS histochemical reaction, the transgene expression pattern had a strong, constitutive character in seven of the transformants. The polyacrylamide gel electrophoresis of a protein extract from the SAK/PCR-positive plants, revealed the presence of a119 kDa protein that corresponds to that of the fusion protein SAK-mGFP-GUSA. Western blot analysis, using an antibody against staphylokinase, showed the presence of the staphylokinase domain in the 119 kDa protein in six analyzed transformants. However, the enzymatic test revealed amidolytic activity characteristic of staphylokinase in the protein extract of only one plant. This is the first report on a Solanum tuberosum plant producing a recombinant staphylokinase protein, a plasminogen activator of bacterial origin.

Expression of a staphylokinase, a thrombolytic agent in Arabidopsis thaliana.

A gene encoding staphylokinase from Staphylococcus aureus was cloned into the plant transformation binary vector pCAMBIA 1304. The transgene was introduced into the genome of A. thaliana via in planta Agrobacterium tumefaciens-mediated genetic transformation. The presence of the staphylokinase gene was confirmed by PCR in 60% of the investigated plants. The presence of the fusion protein (119 kDa) was confirmed by SDS-PAGE and Western blot analysis in protein extracts from putative transgenics. Furthermore, the amidolytic assay confirmed the activity of SAK in protein extracts in 23 out of 45 transgenic lines of A. thaliana plants.

Static magnetic field affects oxidative stress in mouse cochlea.

OBJECTIVE: It has been shown that oxidative stress plays an important role in development of noise induced hearing loss. Since static magnetic fields (SMF) exposure may alter dynamics of oxidative processes in the tissue, the aim of the study was to assess the influence of SMF on noise-induced alteration in the cochlear level of reactive oxygen species (ROS) and hearing thresholds. MATERIALS AND METHODS: Auditory brainstem response (ABR), lipid peroxidation (LPO) levels, super-oxide dismutase (SOD) activity and catalase activity were assessed in the cochlea prior to, and at five time-points over two weeks following exposure of C57BL/6 mice to 8h, 119 dB SPL, 4 kHz octave band noise. RESULTS: The ABR indicated no permanent functional damage due to noise exposure either for the 4 kHz and 8 kHz SMF-exposed group or for animals not exposed to SMF. However, significant differences in LPO level, catalase and SOD activity between animals exposed to noise and SMF and those exposed to noise only were observed. CONCLUSIONS: The results suggest that SMF causes an increase in ROS level in the cochlea after noise exposure and, at the same time, it speeds up activation of antioxidative enzymes.