The Arabidopsis HCC1 gene is a homologue of the copper chaperone SCO1 from the yeast Saccharomyces cerevisiae. SCO1 (synthesis of cytochrome c oxidase 1) encodes a mitochondrial protein that is essential for the correct assembly of complex IV in the respiratory chain. GUS analyses showed HCC1 promoter activity in vascular tissue, guard cells, hydathodes, trichome support cells, and embryos. HCC1 function was studied in two hcc1 T-DNA insertion lines, hcc1-1 and hcc1-2. Gametophyte development was not affected by the disruption of HCC1, but homozygous hcc1-1 and hcc1-2 embryos became arrested at various developmental stages, mostly at the heart stage. Both the wild-type HCC1 gene and the modified gene coding for the C-terminally SNAP-tagged HCC1 were able to complement the embryo-lethal phenotype of the hcc1-1 line. Localization of the SNAP-tagged HCC1 in transgenic lines identified HCC1 as a mitochondrial protein. To determine if HCC1 is a functional homologue to Sco1p, the respiratory-deficient yeast sco1 mutant was transformed with chimeric constructs containing different combinations of HCC1 and SCO1 sequences. One of the resulting chimeric proteins restored respiration in the yeast mutant. This protein had the N-terminal mitochondrial targeting signal and the single transmembrane domain derived from Sco1p and the C-terminal half (including the copper-binding motif) derived from HCC1. Growth of the complemented yeast mutant was enhanced by the addition of copper to the medium. The data demonstrate that HCC1 is essential for embryo development in Arabidopsis, possibly due to its role in cytochrome c oxidase assembly.
Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.
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