Two Arabidopsis thaliana genes (HCC1 and HCC2), resulting from a duplication that took place before the emergence of flowering plants, encode proteins with homology to the SCO proteins involved in copper insertion during cytochrome c oxidase (COX) assembly in other organisms. Heterozygote HCC1 mutant plants produce 25% abnormal seeds with defective embryos arrested at the heart or torpedo stage. These embryos lack COX activity, suggesting that the requirement of HCC1 during the early stages of plant development is related with its COX assembly function. Homozygote HCC2 mutant plants develop normally and do not show changes in COX2 levels. These plants display increased sensitivity of root growth to increased copper and a higher expression of miR398 and other genes that respond to copper limitation, in spite of the fact that they have a higher copper content than the wild type. HCC2 mutant plants also show increased expression of stress-responsive genes. The results suggest that HCC1 is the protein involved in COX biogenesis and that HCC2, that lacks the cysteines and histidine putatively involved in copper binding, functions in copper sensing and redox homeostasis. In addition, plants that overexpress HCC1 have an altered response of root elongation to changes in copper in the growth medium and increased expression of two low-copper-responsive genes, suggesting that HCC1 may also have a role in copper homeostasis.

SummaryPublicly available microarray experiments were used to analyze Arabidopsis thaliana genes whose expression is correlated with that of nuclear genes encoding components of the oxidative phosphorylation machinery (OxPhos genes). This analysis indicated the existence of coordination in the expression of genes encoding components of the five respiratory complexes. For these genes, preferential expression was observed in anthers and roots, especially in the elongation zone, while reduced or very low relative expression was evident in leaves and mature pollen grains. A global induction of OxPhos genes by carbohydrates, photo-destruction of chloroplasts, inhibition of cellulose synthesis, release from dormancy and germination, among other conditions, was also observed. Cluster analysis of the response of Arabidopsis genes to a set of 15 treatments allowed the identification of DNA motifs, known as site II, that are frequently present in the upstream regions of genes with responses like those of OxPhos genes. Mutagenic analysis of site II motifs in several genes encoding respiratory chain components showed that they actively participate in transcription of these genes. We conclude that an important number of nuclear genes encoding components of the five respiratory complexes show coordinated expression under various circumstances, and that site II elements and the putative proteins that interact with them are, together with as yet unidentified factors, important actors in this coordinated response.

AtCOX17 genes encode Arabidopsis thaliana homologs of the yeast metallochaperone Cox17p, involved in the delivery of copper for cytochrome c oxidase (COX) assembly. Two different AtCOX17 genes, located in chromosomes 1 and 3, are present in the Arabidopsis genome. Sequences available in data banks indicate that the presence of two genes is a common feature in monocots, but not in dicots, suggesting that Arabidopsis genes may be the result of a recent duplication. Sequences upstream from the translation start sites of AtCOX17 genes, which include an intron located in the 5′ leader region, were introduced into plants in front of the gus gene. For both genes, expression was localized preferentially in young roots and anthers, but almost 10‐fold higher β‐glucuronidase activity levels were observed in plants transformed with AtCOX17‐1 upstream regions. Both promoters were induced to different extents by wounding, treatment of leaves with the bacterial pathogen Pseudomonas syringae and incubation with agents that produce oxidative stress and metals. AtCOX17‐2 showed similar responses to these factors, while AtCOX17‐1 was more strongly induced by relatively low (10–100 μM) copper. The results indicate that both AtCOX17 genes have similar, though not identical, expression characteristics and suggest the existence in their promoters of elements involved in tissue‐specific expression and in responses to factors that may produce mitochondrial or cell damage. It can be speculated that Arabidopsis COX17 accumulates under stress conditions to actively replace damaged or inactive cytochrome c oxidase to sustain cyanide‐sensitive respiration in plant cells.

The Arabidopsis thaliana genome contains two nearly identical genes which encode proteins showing similarity with the yeast metal chaperone Cox19p, involved in cytochrome c oxidase biogenesis. One of these genes (AtCOX19-1) produces two transcript forms that arise from an alternative splicing event and encode proteins with different N-terminal portions. Both AtCOX19 isoforms are imported into mitochondria in vitro and are found attached to the inner membrane facing the intermembrane space. The smaller AtCOX19-1 isoform, but not the larger one, is able to restore growth on non-fermentable carbon sources when expressed in a yeast cox19 null mutant. AtCOX19 transcript levels increase by treatment with copper or compounds that produce reactive oxygen species. Young roots and anthers are highly stained in AtCOX19-1::GUS plants. Expression in leaves is only observed when cuts are produced, suggesting an induction by wounding. Infection of plants with the pathogenic bacterium Pseudomonas syringae pv. tomato also induces AtCOX19 gene expression. The results suggest that AtCOX19 genes encode functional homologues of the yeast metal chaperone. Induction by biotic and abiotic stress factors may indicate a relevant role of this protein in the biogenesis of cytochrome c oxidase to replace damaged forms of the enzyme or a more general role in the response of plants to stress.

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Herein, a microfluidic device with cistern design for cultivation of adherent eukaryotic cells for the production of recombinant proteins is presented. The geometric configuration of the microchannels in the device provided laminar flow with reduced velocity profiles in the cisterns, resulting in an adequate microenvironment for long-term adherent cell growth with passive pumping flow cycles of 24 hours. CHO-ahIFNα2b and HEK-ahIFNα2b adherent cell lines expressing a novel anti-hIFN-α2b recombinant monoclonal antibody (MAb) for the treatment of systemic lupus erythematosus were cultured on the surface of PDMS/glass microchannels coated with poly-d-lysine. A 24 day culture of CHO-ahIFNα2b cells resulted in MAb concentrations up to 166.4 μg mL per day. The productivity of CHO-ahIFNα2b and HEK-ahIFNα2b cell lines was higher in the microdevice compared to that obtained using the adherent cell culture method (T-flask), with a 5.89- and 7.31-fold increase, respectively. Moreover, biological analysis of the MAbs produced in the microdevice showed no significant differences in the neutralizing antiproliferative activity of the hIFN-α2b or the cytokine cell signaling compared to the MAbs produced with cell adherent methods. These results suggest that this microfluidic device is suitable for long-term culture of mammalian cells and can improve the productivity of cells expressing recombinant MAbs with potential for therapeutic use without affecting the quality attributes of the product.

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