Plant cell cultures: Chemical factories of secondary metabolites

2. Evidence for A m i n o Acids as T ransmi t t e r s . . . . . . . . . . . . . . . . . . . . 99 2.1. Synthesis and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 2.2. Synapt ic Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 2.3. Postsynapt ic Act ion . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 2.4. Postsynapt ic An tagon i s t s . . . . . . . . . . . . . . . . . . . . . . . . . 103 2.5. Inac t iva t ion and R e m o v a l . . . . . . . . . . . . . . . . . . . . . . . . . 104

Amino acid transmitters in the mammalian central nervous system

Imagine a world in which any protein, either naturally occurring or designed by man, could be produced safely, inexpensively and in almost unlimited quantities using only simple nutrients, water and sunlight. This could one day become reality as we learn to harness the power of plants for the production of recombinant proteins on an agricultural scale. Molecular farming in plants has already proven to be a successful way of producing a range of technical proteins. The first plant-derived recombinant pharmaceutical proteins are now approaching commercial approval, and many more are expected to follow.

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The production of recombinant pharmaceutical proteins in plants.

Plants and human health in the twenty-first century

Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants

Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a nominally constitutive promoter. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed the presence of HBsAg in extracts of transformed leaves at levels that correlated with mRNA abundance. This suggests that there were no major inherent limitations of transcription or translation of this foreign gene in plants. Recombinant HBsAg was purified from transgenic plants by immunoaffinity chromatography and examined by electron microscopy. Spherical particles with an average diameter of 22 nm were observed in negatively stained preparations. Sedimentation of transgenic plant extracts in sucrose and cesium chloride density gradients showed that the recombinant HBsAg and human serum-derived HBsAg had similar physical properties. Because the HBsAg produced in transgenic plants is antigenically and physically similar to the HBsAg particles derived from human serum and recombinant yeast, which are used as vaccines, we conclude that transgenic plants hold promise as low-cost vaccine production systems.

https://www.pnas.org/content/pnas/89/24/11745.full.pdf

Expression of hepatitis B surface antigen in transgenic plants.

After goldfish retinas had been incubated for 1 hr with [γ-3H]aminobutyric acid, we found by autoradiography that the label was localized to a few restricted types of retinal cells. In particular, external and internal horizontal cells from light-stimulated retinas were more heavily labeled than corresponding cells from retinas kept in darkness. Some other cells and tissues in the retina also incorporated the labeled acid. Light stimulation, however, did not cause a pronounced change in the amount of label associated with these cells. Among these were some heavily labeled cells on the vitreal side of the inner nuclear layer, and scattered grains associated with the ganglion cell and optic nerve layers. Electrophoresis of retinal extracts after incubation with the labeled acid also showed that light-stimulated retinas contained about 40-100% more radioactivity than retinas kept in darkness, and that 90% of this activity remained as [γ-3H]aminobutyric acid. The role of the acid in the retina is not known; it is not clear if horizontal cells normally synthesize or store it. The stimulation-dependent accumulation of the labeled acid into horizontal cells suggests that it plays a functional role in these cells.

https://www.pnas.org/content/pnas/68/11/2777.full.pdf

The Uptake of [γ-3H] Aminobutyric Acid in the Goldfish Retina

The anti-viral vaccine of the present invention is produced in transgenic plants and then administered through standard vaccine introduction method or through the consumption of the edible portion of those plants. A DNA sequence encoding for the expression of a surface antigen of a viral pathogen is isolated and ligated to a promoter which can regulate the production of the surface antigen in a transgenic plant. This gene is then transferred to plant cells using a procedure that results in its integration into the plant genome, such as through the use of an Agrobacterium tumenfaciens plasmid vector system. Preferably, the foreign gene is expressed in an portion of the plant that is edible by humans or animals. In a preferred procedure, the vaccine is administered through the consumption of the edible plant as food, preferably in the form of a fruit or vegetable juice which can be taken orally.

Vaccines expressed in plants

For determination of possible neurotransmitters synthesized by photoreceptor cells, turtle retinas were dissociated into single cells with proteolytic enzymes. These cells were partially separated by velocity sedimentation to yield a fraction rich in photoreceptors. Individual photoreceptor cells were then sucked into a micropipette and incubated with labeled precursors of known or suspected neurotransmitters. After incubation, the radioactive products were analyzed by high-voltage electrophoresis. Of all the chemicals tested, turtle photoreceptor cells synthesized only acetylcholine, suggesting that these cells may be cholinergic.

Biosynthesis of Acetylcholine in Turtle Photoreceptors

In the catfish retina, horizontal cells that receive inputs exclusively from red-sensitive cones are the only neurons that accumulate exogenous gamma-aminobutyric acid under our experimental conditions. When isolated eyecups are perfused with bicuculline methochloride, an antagonist of postsynaptic gamma-aminobutyric acid receptors, responses of cone photoreceptors to a field of light (3 mm in diameter) become much slower and approach those to a small spot of light (0.3 mm). In addition, bicuculline methochloride decreases the frequency responses of cone horizontal cells to a field of light. These findings indicate that, in the catfish retina, feedback synapses from cone horizontal cells to cones are chemically mediated and may use gamma-aminobutyric acid as a neurotransmitter. Our results also confirm the hypothesis that, in the catfish retina, a function of the negative feedback is to improve the frequency responses of the system.

https://www.pnas.org/content/pnas/75/12/6310.full.pdf

Dominic Man-Kit Lam

Papers

Expression of hepatitis B surface antigen in transgenic plants.

The Uptake of [γ-3H] Aminobutyric Acid in the Goldfish Retina

Vaccines expressed in plants

Biosynthesis of Acetylcholine in Turtle Photoreceptors

gamma-Aminobutyric acid: a neurotransmitter candidate for cone horizontal cells of the catfish retina.