Production of recombinant single chain antibodies (scFv) in vegetatively reproductive Kalanchoe pinnata by in planta transformation.

We developed an asexual reproductive plant, Kalanchoe pinnata, as a new bioreactor for plant-based molecular farming using a newly developed transformation method. Leaf crenate margins were pin-pricked to infect the plant with the Agrobacterium strain LBA4404 and vacuum infiltration was also applied to introduce the target gene into the plants. Subsequently, the young mother leaf produced new clones at the leaf crenate margins without the need for time- and labor-consuming tissue culture procedures. The average transformation rates were approximately 77 and 84% for pin-prickling and vacuum-infiltration methods, respectively. To functionally characterize an introduced target protein, a nucleic acid hydrolyzing recombinant 3D8 scFv was selected and the plant based 3D8 scFv proteins were purified and analyzed. Based on abzyme analysis, the purified protein expressed with this system had catalytic activity and exhibited all of properties of the protein produced in an E. coli system. This result suggested that vegetatively reproductive K. pinnata can be a novel and potent bioreactor for bio-pharmaceutical proteins.

Hydrogen peroxide induces the death of astrocytes through the down-regulation of the constitutive nuclear factor-kappaB activity.

Nuclear factor-kappaB (NF-kappaB) has a dual role in the promotion or attenuation of cell death. Here, we demonstrated the role of NF-kappaB in the H(2)O(2)-induced death of astrocytes. H(2)O(2) evoked the release of lactate dehydrogenase (LDH), a marker of cell death, and concomitantly decreased the DNA binding and transcriptional activity of NF-kappaB in cultured astrocytes. H(2)O(2)-induced astrocyte death was markedly increased by the co-treatment with pyrrolidinedithiocarbamate, an NF-kappaB inhibitor. Moreover, the elevation of constitutive NF-kappaB activity by overexpressing p65 NF-kappaB subunit attenuated H(2)O(2) toxicity, whereas NF-kappaB inhibition by overexpressing IkappaB potentiated the toxicity. NF-kappaB activity and H(2)O(2) cytotoxicity was further found to be dependent on cell density. Compared with astrocytes in low cell density, those in high cell density exhibited a higher constitutive NF-kappaB activity and a stronger resistance to H(2)O(2) cytotoxicity. These results indicate that the constitutive activity of NF-kappaB in astrocytes is required for their survival under oxidative stress such as H(2)O(2).

Mimosine prevents the death of glucose-deprived immunostimulated astrocytes by scavenging peroxynitrite.

Immunostimulated astrocytes become highly vulnerable to glucose deprivation (Choi and Kim: J Neurosci Res 54:870-875, 1998a). The increased vulnerability is caused by the enhanced level of peroxynitrite endogenously produced in glucose-deprived immunostimulated astrocytes. In the present study, we report that the plant amino acid mimosine can attenuate the increased death by scavenging peroxynitrite. Treatment with mimosine blocked the increase of nitrotyrosine immunoreactivity, a marker of peroxynitrite, in glucose-deprived immunostimulated astrocytes. Furthermore, mimosine directly inhibited the nitration of tyrosine residues of bovine serum albumin and the oxidation of dihydrorhodamine-123 to rhodamine-123 by peroxynitrite. Mimosine has been used experimentally as a cell cycle G1/S phase transition blocker (Lalande: Exp Cell Res 186:332-339, 1990; Hoffman et al.: Cytometry 12:26-32, 1991). Flow cytometry analysis, however, showed that the cytoprotective effect of mimosine was not attributed to its inhibition of cell cycle progression. Furthermore, under our experimental conditions, mimosine did not alter the levels of cell cycle regulatory proteins, including p21(WAF1/CIP1), cyclins D1 and E, and proliferating cell nuclear antigen. In addition, cyclin-dependent kinase inhibitors olomoucine and roscovitine did not block the increased death. These results indicate that mimosine inhibits the augmented death of glucose-deprived immunostimulated astrocytes by scavenging peroxynitrite rather than suppressing the cell cycle progression.

Glucose deprivation decreases nitric oxide production via NADPH depletion in immunostimulated rat primary astrocytes.

We have previously reported that the production of nitric oxide (NO) in immunostimulated astrocytes was markedly decreased under glucose-deprived conditions. The present study was undertaken to find the contributing factor(s) for the decreased NO production in glucose-deprived immunostimulated astrocytes. NO production in rat primary astrocytes was stimulated for 24-48 h by cotreatment with lipopolysaccharides (1 microg/ml) and interferon-gamma (100 U/ml). Decreased NO production in immunostimulated astrocytes by glucose deprivation was mimicked by the glycolytic inhibitor 2-deoxyglucose and reversed by addition of pyruvate and lactate. Glucose deprivation did not alter the expression of inducible nitric oxide synthase (iNOS) in immunostimulated astrocytes. Addition of beta-NADPH, but not tetrahydrobiopterine, both of which are essential cofactors for NOS function, completely restored the NO production that was decreased in glucose-deprived immunostimulated astrocytes. Glucose deprivation and immunostimulation synergistically reduced intracellular NADPH level in astrocytes. The results indicate that glucose deprivation decreases NO production in immunostimulated astrocytes by depleting intracellular NADPH, a cofactor of iNOS.