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Violaxanthin de-epoxidase and zeaxanthin epoxidase catalyze the addition and removal of epoxide groups in carotenoids of the xanthophyll cycle in plants. The xanthophyll cycle is implicated in protecting the photosynthetic apparatus from excessive light. Two new sequences for violaxanthin de-epoxidase from tobacco and Arabidopsis are described. Although the mature proteins are well conserved, the transit peptides of these proteins are divergent, in contrast to transit peptides from other proteins targeted to the thylakoid lumen. Sequence analyses of both violaxanthin de-epoxidase and zeaxanthin epoxidase establish the xanthophyll cycle enzymes as members of the lipocalin family of proteins. The lipocalin family is a diverse group of proteins that bind small hydrophobic (lipophilic) molecules and share a conserved tertiary structure of eight ␤-strands forming a barrel configuration. This is the first reported identification of lipocalin proteins in plants.The xanthophyll cycle is comprised of de-epoxidation and epoxidation interconversions of three xanthophylls (violaxanthin, antheraxanthin, and zeaxanthin), catalyzed by two enzymes that are localized on opposite sides of the thylakoid membrane. Violaxanthin de-epoxidase (VDE) 1 is localized in the lumen of thylakoids and catalyzes de-epoxidation of violaxanthin in the presence of ascorbate and an acidic lumen, the latter formed by the light-induced proton pump (1-5). Zeaxanthin epoxidase is localized on the stromal side of the thylakoid membrane and catalyzes the epoxidation of zeaxanthin in the dark or under low light intensities (6, 7). The reaction is optimal near pH 7.5, and the enzyme utilizes oxygen, ferredoxin, and FAD as co-substrates (6 -10). A role for zeaxanthin was first described in 1987 by Demmig et al. (11) to protect the photosynthetic apparatus against the adverse effects of excessive light. Recent evidence demonstrates that accumulation of both antheraxanthin and zeaxanthin, along with the transthylakoid pH gradient, mediates the non-radiative dissipation of excess energy as heat (12)(13)(14)(15)(16)(17). The xanthophyll cycle is thought to have evolved early in the development of higher plants as it is present in all plants examined thus far (18). Pervaiz and Brew (19,20) first identified a group of proteins, based on sequence homology, that have a common role in binding and transport of small hydrophobic molecules. These proteins, designated the lipocalins, represent a diverse group of proteins from the animal kingdom (for review see Ref. 21) and recently from a prokaryote (22). These lipocalin proteins have a common tertiary structure of an eight-stranded anti-parallel ␤-barrel, and only one protein to date displays catalytic activity. We report that violaxanthin de-epoxidase and zeaxanthin epoxidase are members of the lipocalin family. To our knowledge, they are the first lipocalins described from plants and only the second reported to demonstrate enzymatic activity. The deduced polypeptide sequences of three VDE proteins are compared, a...

The gene (PRAII) encoding a secreted aspartate proteinase of Candida albicans has been cloned and sequenced. The nucleotide and deduced amino acid sequences ofPRAII are 77 and 73% identical, respectively, with the reported sequences of PRAIO also cloned from C. albicans. Southern analyses indicated that the genome of each strain examined (ATCC 10231 and ATCC 10261) contains PRAIO and PRAII. Northern (RNA) analyses showed that PRAII was expressed at a much higher level than was PRA10 when secretion of the proteinase by strain ATCC 10261 was induced with albumin.The opportunistic pathogen Candida albicans secretes an aspartate proteinase (EC 3.4.23.6) Enzyme purification and protein analysis. Ten-liter cultures of strain ATCC 10261 were grown in a medium containing 0.2% yeast extract, 0.2% bovine serum albumin, and 2% (wt/vol) glucose (12 h at 30°C with aeration at 10 liters min-1 and a speed of 400 rpm). After cell harvesting, the medium was adjusted from pH 3.0 to pH 7.0 with 6 M NaOH and concentrated to 400 to 500 ml by ultrafiltration (10,000 molecular weight cutoff). After dialysis for 12 h against 10 mM sodium citrate buffer (pH 6.8), the concentrate was applied to a DEAE-Sephacel column (26 by 2.5 cm) equilibrated with the same buffer, and the column was washed

Loss of gap junctional intercellular communication (GJIC) has been linked to aberrant proliferation and an enhanced neoplastic phenotype. Many human tumors, including the cervical carcinoma line HeLa, have been reported to be deficient in expression of the gap junction protein connexin43 (Cx43) and GJIC. To determine if this is an early event in carcinogenesis, we utilized immunohistochemistry to screen a series of cervical biopsy samples and demonstrated a major reduction in Cx43 expression in dysplastic regions compared to normal epithelia. To determine whether this loss influences the neoplastic behavior of cervical carcinoma cells, we have constructed HeLa cell lines in which Cx43 expression can be induced in response to doxycycline. This approach allows for the discrimination of Cx43-mediated effects from those due to pre-existing clonal heterogeneity. Cx43 induction in these cells led to assembly of functional junctions but did not alter growth control in vitro as measured by logarithmic growth, saturation density or focus formation when in co-culture with growth-controlled fibroblasts. However, Cx43 induction decreased two indices of neoplasia: it reduced anchorage-independent growth and attenuated the growth rate of tumor xenografts. These results indicate that established HeLa cell lines are unresponsive to Cx43-mediated signals which are thought to mediate growth control of non-transformed cells, however, Cx43 expression can still reduce aspects of the neoplastic phenotype of these cells, indicating that loss of connexin signaling in dysplastic cells may contribute to their neoplastic progression.

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Orchids are one of the most unique and evolved of flowering plants, with many being valuable floricultural crops. Spatial localization of pigments within the flower of the commercially important bi-color Oncidium Gower Ramsey demonstrated a mixture of carotenoids and anthocyanins concentrated in the adaxial epidermis. Chromatography identified the predominant yellow pigment to be an equal mixture of all-trans and 9-cis isomers of violaxanthin, with esterification specific to the 9-cis isomer. Red ornamentation was comprised of the anthocyanins cyanidin and its methylated derivate, peonidin. Five key pigment biosynthesis genes encoding dihydroflavonol 4-reductase (DFR), phytoene synthase (PSY), phytoene desaturase, carotenoid isomerase, and the downstream 9-cis epoxycarotenoid dioxygenase were isolated and their expression profiles determined. Northern analyses showed both phytoene desaturase and carotenoid isomerase expression to be up-regulated in floral tissue relative to leaves whereas PSY was not. Three closely related DFR genes were isolated, including one with an insertion in the 3' coding region. DFR expression occurred throughout flower development in Oncidium, unlike in Dendrobium and Bromheadia orchids. A number of the isolated anthocyanin and carotenoid genes showed variations due to insertion events. These findings raise questions about the genetic stability in interspecific crosses in orchids, such as the tri-specific Oncidium Gower Ramsey.