Study on the mechanism of Bioelectric Recognition Assay: evidence for immobilized cell membrane interactions with viral fragments

The Bioelectric Recognition Assay (BERA) is a whole-cell based biosensing system that detects the electric response of cultured cells, suspended in a gel matrix, to various ligands, which bind to the cell and/or affect its physiology. Previous studies have demonstrated the potential application of this method for rapid, inexpensive detection of viruses in a crude sample. However, the understanding, so far, of the fundamental processes that take place during cell-virus interactions within the probe has been rather limited. In the present study, we combined electrophysiological and fluorescence microscopical assays, so that we can prove that animal and plant cells immobilized in BERA sensors respond to different viruses primarily by changing their membrane potential. The response of immobilized cells against different viruses did not depend on the virus ability to penetrate the cell, but was modified after binding each virus to a virus-specific antibody or removal of its coat protein after treatment with a protease. Consequently, we were able to assay the presence of a virus in its complete form or fragments thereof. Combination of immunological recognition with the electrophysiological response of immobilized cells allows for a considerable increase of the specificity of the BERA biosensory assay. In addition, rather than simply detect the presence of a protein or genomic sequence, the method can help gain information on the bioactivity of a virus.     

Induction of somatic embryogenesis and in vitro flowering from inflorescences of chamomile (Chamomilla recutita L.)

A protocol has been developed for the induction of somatic embryogenesis from flower explants of chamomile (Chamomilla recutita L.). The effects of several plant growth regulators [α-naphthylacetic acid (NAA), 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA) and kinetin (Kin), alone or in combination] and the flower type (disk or ray flower) were investigated. Both types of flowers responded to the callus and shoot induction treatments, but formation of globular somatic embryos took place only on disk-flower-derived explants after 2–4 weeks of culture on a Murashige and Skoog (MS) medium supplemented either with 8.87 μm BA and 1.07 μm NAA or with 26.8 μm NAA and 11.5 μm Kin. However, fully developed, cotyledonary-stage somatic embryos could be induced only on the NAA/Kin medium, 10 weeks after culture initiation. Germination of the embryos and plant regeneration took place after subculture for 4–5 weeks onto medium of the same composition. Plantlets regenerated from embryos flowered in vitro on a MS medium supplemented with 8.87 μm BA and 1.07 μm NAA. The significance of the results with respect to chamomile micropropagation and the utilization of wild populations in breeding programs is discussed. Received: 6 April 1998 / Revision received: 12 October 1998 / Accepted: 28 October 1998     

Anther culture response of Hordeum spontaneum-derived winter barley lines

The anther culture response of 18 Hordeum spontaneum derived winter barley lines, as well as that of the F₂ and/or BC₁ progeny of the cross between nine of these lines and the winter barley cultivar Triton was assessed. While the majority of lines showed a poor regeneration capacity (4%), two lines, RS 170-A13 × Sonja and RS 120-27 × Sonja demonstrated an outstanding response, producing 26 and 8 green regenerants per 100 anthers plated, respectively. Five further lines, RS 70-30 × Dura, RS L74-11 × Dura, RS 122-19 × Sonja. RS 142-29 × Dura and RS 170-A51 × Sonja were identified as highly responsive in the F₂ generation. An average production of 4 green regenerants per 100 anthers plated was observed both in the parental and F₂ generation.Abbreviations BA benzyladenine - IAA indole-3-acetic acid     

Moderate superoxide production is an early promoter of mitochondrial biogenesis in differentiating N2a neuroblastoma cells

Reactive oxygen species (ROS) have been widely considered as harmful for cell development and as promoters of cell aging by increasing oxidative stress. However, ROS have an important role in cell signaling and they have been demonstrated to be beneficial by triggering hormetic signals, which could protect the organism from later insults. In the present study, N2a murine neuroblastoma cells were used as a paradigm of cell-specific (neural) differentiation partly mediated by ROS. Differentiation was triggered by the established treatments of serum starvation, forskolin or dibutyryl cyclic AMP. A marked differentiation, expressed as the development of neurites, was detected by fixation and staining with coomassie brilliant blue after 48 h treatment. This was accompanied by an increase in mitochondrial mass detected by mitotracker green staining, an increased expression of the peroxisome proliferator-activated receptor gamma (PPARγ) coactivator 1-alpha (PGC-1α) and succinate dehydrogenase activity as detected by MTT. In line with these results, an increase in free radicals, specifically superoxide anion, was detected in differentiating cells by flow cytometry. Superoxide scavenging by MnTBAP and MAPK inhibition by PD98059 partially reversed differentiation and mitochondrial biogenesis. In this way, we demonstrated that mitochondrial biogenesis and differentiation are mediated by superoxide and MAPK cues. Our data suggest that differentiation and mitochondrial biogenesis in N2a cells are part of a hormetic response which is triggered by a modest increase of superoxide anion concentration within the mitochondria.     

Differential effect of the shape of calcium alginate matrices on the physiology of immobilized neuroblastoma N2a and Vero cells: a comparative study

In order to investigate the effect of cell immobilization in calcium alginate gels on cell physiology, we immobilized Vero or N2a neuroblastoma cells in gels shaped either as spherical beads or as thin membrane layers. Throughout a culture period of 4 weeks cell viability, RNA and cytoplasmic calcium concentration and glutathione accumulation were assayed by fluorescence microscopy after provision of an appropriate dye. Non-elaborate culture conditions were applied throughout the experimental period in order to evaluate cell viability under less than optimal storage conditions. Vero cell proliferation was observed only in spherical beads, while N2a cell proliferation was observed in both configurations until the third week of culture. Increased [Ca2+]cyt could be associated with cell proliferation only when cells were immobilized in spherical beads, while a considerable decrease in the biosynthesis of reduced glutathione and RNA was observed in cells immobilized in thin membrane layers. The observed effects of the shape of the immobilization matrix may be due to differences in external mass transfer resistance. Therefore, depending on cell type, cell proliferation could have been promoted by either increased (Vero) or decreased (N2a) nutrient and oxygen flow to immobilized cells. The results of the present study could contribute to an improvement of immobilized cell sensor storability.     

Engineering of the membrane of fibroblast cells with virus-specific antibodies: A novel biosensor tool for virus detection

A novel concept for the assay of viral antigens is described. The methodological approach is based on a membrane-engineering process involving the electroinsertion of virus-specific antibodies in the membranes of fibroblast cells. As a representative example, Vero fibroblasts were engineered with antibodies against Cucumber mosaic virus (CMV) and used for the construction of an ultra-sensitive miniature cell biosensor system. The attachment of a homologous virus triggered specific changes to the cell membrane potential that were measured by appropriate microelectrodes, according to the principle of the bioelectric recognition assay (BERA). No change in the membrane potential was observed upon cell contact with the heterologous cucumber green mottle mosaic virus (CGMMV). Fluorescence microscopy observations showed that attachment of CMV particles to membrane-engineered cells was associated with membrane hyperpolarization and increased [Ca(2+)](cyt). In an additional field-based application, we were able to detect CMV-infected tobacco plants at an essentially 100% level of accuracy.     

Visualization of the membrane engineering concept: evidence for the specific orientation of electroinserted antibodies and selective binding of target analytes

Membrane engineering is a generic methodology for increasing the selectivity of a cell biosensor against a target molecule, by electroinserting target‐specific receptor‐like molecules on the cell surface. Previous studies have elucidated the biochemical aspects of the interaction between various analytes (including viruses) and their homologous membrane‐engineered cells. In the present study, purified anti‐biotin antibodies from a rabbit antiserum along with in‐house prepared biotinylated bovine serum albumin (BSA) were used as a model antibody‐antigen pair of molecules for facilitating membrane engineering experiments. It was proven, with the aid of fluorescence microscopy, that (i) membrane‐engineered cells incorporated the specific antibodies in the correct orientation and that (ii) the inserted antibodies are selectively interacting with the homologous target molecules. This is the first time the actual working concept of membrane engineering has been visualized, thus providing a final proof of the concept behind this innovative process. In addition, the fluorescence microscopy measurements were highly correlated with bioelectric measurements done with the aid of a bioelectric recognition assay. Copyright © 2013 John Wiley & Sons, Ltd.     

Scale-up micropropagation of sweet basil (Ocimum basilicum L.) in an airlift bioreactor and accumulation of rosmarinic acid

Nodal explants with lateral buds and leaf-derived suspension cultures of sweet basil, Ocimum basilicum L., were cultured in 5 l airlift bioreactors for three weeks, thereby increasing the fresh wt of suspension cultures 2.5-fold. Rosmarinic acid accumulated at 29 micromicrog g(-1) dry wt in the suspension culture but, for micropropagated plants, it reached 178 microg g(-1) dry wt.     

Morpho-histological study on shoot bud regeneration in cotyledon cultures of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

Adventitious organogenetic structures were produced in vitro using cotyledon explants excised from 14 day-old pepper seedlings. The best response was observed on MS medium containing 5.7 μM indole-3-acetic acid and 8.8 μM 6-benzylaminopurine. However, when transferred onto elongation medium (MS + 2.8 μM gibberellic acid), these structures frequently developed into leaf-like features rather than into normal shoots. Interestingly, the histological study conducted on the cut end of the cotyledonary petiole revealed a direct induction of numerous teratological protuberances that arise around the cut end of the explant. On the contrary, typical organized bud meristems were rarely observed. Thus, the low number of plants obtained after transfer onto elongation medium seems to be a consequence of this teratological developmental process, frequently associated with fasciated and degenerative meristems, rather than a defect in shoot development from available meristems.