Homology models of human all-trans retinoic acid metabolizing enzymes CYP26B1 and CYP26B1 spliced variant.

Homology models of CYP26B1 (cytochrome P450RAI2) and CYP26B1 spliced variant were derived using the crystal structure of cyanobacterial CYP120A1 as template for the model building. The quality of the homology models generated were carefully evaluated, and the natural substrate all-trans-retinoic acid (atRA), several tetralone-derived retinoic acid metabolizing blocking agents (RAMBAs), and a well-known potent inhibitor of CYP26B1 (R115866) were docked into the homology model of full-length cytochrome P450 26B1. The results show that in the model of the full-length CYP26B1, the protein is capable of distinguishing between the natural substrate (atRA), R115866, and the tetralone derivatives. The spliced variant of CYP26B1 model displays a reduced affinity for atRA compared to the full-length enzyme, in accordance with recently described experimental information.

Cloning and functional studies of a splice variant of CYP26B1 expressed in vascular cells.

BACKGROUND: All-trans retinoic acid (atRA) plays an essential role in the regulation of gene expression, cell growth and differentiation and is also important for normal cardiovascular development but may in turn be involved in cardiovascular diseases, i.e. atherosclerosis and restenosis. The cellular atRA levels are under strict control involving several cytochromes P450 isoforms (CYPs). CYP26 may be the most important regulator of atRA catabolism in vascular cells. The present study describes the molecular cloning, characterization and function of atRA-induced expression of a spliced variant of the CYP26B1 gene. METHODOLOGY/PRINCIPAL FINDINGS: The coding region of the spliced CYP26B1 lacking exon 2 was amplified from cDNA synthesized from atRA-treated human aortic smooth muscle cells and sequenced. Both the spliced variant and full length CYP26B1 was found to be expressed in cultured human endothelial and smooth muscle cells, and in normal and atherosclerotic vessel. atRA induced both variants of CYP26B1 in cultured vascular cells. Furthermore, the levels of spliced mRNA transcript were 4.5 times higher in the atherosclerotic lesion compared to normal arteries and the expression in the lesions was increased 20-fold upon atRA treatment. The spliced CYP26B1 still has the capability to degrade atRA, but at an initial rate one-third that of the corresponding full length enzyme. Transfection of COS-1 and THP-1 cells with the CYP26B1 spliced variant indicated either an increase or a decrease in the catabolism of atRA, probably depending on the expression of other atRA catabolizing enzymes in the cells. CONCLUSIONS/SIGNIFICANCE: Vascular cells express the spliced variant of CYP26B1 lacking exon 2 and it is also increased in atherosclerotic lesions. The spliced variant displays a slower and reduced degradation of atRA as compared to the full-length enzyme. Further studies are needed, however, to clarify the substrate specificity and role of the CYP26B1 splice variant in health and disease.

The role of the pyridoxine (vitamin B6) biosynthesis enzyme PDX1 in ultraviolet-B radiation responses in plants.

Ultraviolet-B radiation regulates plant growth and morphology at low and ambient fluence rates but can severely impact on plants at higher doses. Some plant UV-B responses are related to the formation of reactive oxygen species (ROS) and pyridoxine (vitamin B(6)) has been reported to be a quencher of ROS. UV-B irradiation of Arabidopsis Col-0 plants resulted in increased levels of PDX1 protein, compared with UV-A-exposed plants. This was shown by immunoblot analysis using specific polyclonal antibodies raised against the recombinant PDX1.3 protein and confirmed by mass spectrometry analysis of immunoprecipitated PDX1. The protein was located mainly in the cytosol but also to a small extent in the membrane fraction of plant leaves. Immunohistochemical analysis performed in pea revealed that PDX1 is present in UV-B-exposed leaf mesophyll and palisade parenchyma but not in epidermal cells. Pyridoxine production increased in Col-0 plants exposed to 3 days of UV-B, whereas in an Arabidopsis pdx1.3 mutant UV-B did not induce pyridoxine biosynthesis. In gene expression studies performed after UV-B exposure, the pdx1.3 mutant showed elevated transcript levels for the LHCB1*3 gene (encoding a chlorophyll a/b-binding protein of the photosystem II light-harvesting antenna complex) and the pathogenesis-related protein 5 (PR-5) gene, compared with wild type.

Production of the p24 capsid protein from HIV-1 subtype C in Arabidopsis thaliana and Daucus carota using an endoplasmic reticulum-directing SEKDEL sequence in protein expression constructs.

An optimized gene expression construct was designed in order to increase the accumulation of the HIV-1 subtype C p24 protein in Arabidopsis thaliana and carrot (Daucus carota) plants. An ER retention signal was introduced into the genetic construct generating a p24 protein containing a SEKDEL amino acid sequence at its C-terminus. Mature A. thaliana plants and carrot cells were transformed using Agrobacterium tumefaciens carrying the improved pGreen0229/p24_SEKDEL vector. Several transgenic plant lines were obtained from both plant species by growth on selective medium and confirmed by PCR. Transformed lines were analyzed for p24 protein content by western blotting using anti-p24-specific antibodies and by Southern blotting to establish the number of copies of the insert in the plant nuclear genome. To estimate the accumulation levels of p24 protein in the plants, ELISA was run using soluble plant extracts. By comparing these results with our previous findings, the ER retention signal increased the level of p24 protein fivefold in the A. thaliana plants. In carrot taproot, the content of p24_SEKDEL protein was approximately half of that in Arabidopsis on a fresh weight basis and was stable in planta for several months. However, on a total soluble protein basis, carrots produced considerable higher levels of the p24_SEKDEL protein than Arabidopsis.

Feeding of mice with Arabidopsis thaliana expressing the HIV-1 subtype C p24 antigen gives rise to systemic immune responses.

Development of transgenic edible plants, to be used as production, storage and delivery systems for recombinant vaccine antigens, is a promising strategy to obtain cost effective vaccines against infectious diseases, not least for use in developing countries. Therefore, we used Agrobacterium tumefaciens-mediated gene transfer to introduce the p24 gag gene encoding the nucleocapsid protein from HIV-1 subtype C into the Arabidopsis thaliana plant genome. Eighteen plant lines were confirmed positive for the p24 gene by PCR; four of these lines showed an apparent homozygous phenotype when grown on selective medium and these lines also showed transcription of the p24 gene into its corresponding mRNA. The mRNA in all four cases generated the p24 protein in plants, as verified by Western blot analysis. The plants were shown to contain between 0.2 mug and 0.5 mug p24 protein per g of fresh tissue. Analysis of the localisation of the p24 protein showed that stem tissue contained the largest amount of protein, more than twice as much as leaf tissue, whereas no p24 protein was detected in roots. By using Southern blotting, we found that 4, 2-3, 2 and 1 T-DNA insertion events took place in the four lines 1, 2, 7, and 10, respectively. The genetic insertions of line 1 were stable from the T(2) to the T(5) generation and gave rise to the p24 protein in all cases, as verified by Western blotting. In mice fed with fresh transgenic A. thaliana (line 10), anti-gag IgG was obtained in serum after a booster injection with recombinant p37Gag. No immune response was observed after equal booster injection of untreated mice or mice fed with A. thaliana WT plants.

A Pisum sativum glyoxysomal malate dehydrogenase induced by cadmium exposure.

The glyoxysomal malate dehydrogenase (gMDH) catalyses the formation of oxaloacetate from malate during beta-oxidation of fatty acids in the glyoxysome. A partial Pisum sativum L. (cv. Greenfeast) cDNA was first isolated from a suppression subtractive hybridisation cDNA library obtained from heavy metal stressed plants. The full length cDNA was then isolated by rapid amplification of cDNA ends. The translated sequence showed strong similarity to Cucumis sativus and Citrullus lanatus gMDH including a typical glyoxysome-targeting presequence comprising the PTS2 motif and a cleavage site for a cystein-directed protease. Exposure of pea plants to Cd2+ induced expression of the gMDH gene in mature pea leaves indicating that the enzyme is under environmental control in addition to the normal developmental regulation pattern.

Ultraviolet-B signalling: Arabidopsis brassinosteroid mutants are defective in UV-B regulated defence gene expression.

The involvement of brassinosteroids in signalling events in plants during UV-B stress (280-315 nm) was investigated in Arabidopsis thaliana. Brassinosteroids are involved in growth and development in plants and have also been shown to enhance stress tolerance. Three mutants deficient in the biosynthetic pathway of brassinolide (BL; det2, dim1 and cpd) and the BL insensitive mutant (bri1) were together with visible light irradiated with 3 or 9 h of UV-B radiation (biologically effective radiation normalised to 300 nm being 0.24 W m(-2)). Also, a small size control, irx1, and Columbia 0 (Col-0) wild-type plants were examined under identical conditions. Gene expression patterns were established for these mutants with a set of four molecular markers (the defence genes chalcone synthase (CHS), PYROA, pathogenesis-related protein PR-5, and a gene regulated by very low levels of UV-B, MEB5.2). Although the genes in the brassinodefective mutants were still induced by UV-B radiation, they all also showed reduced levels of mRNA transcripts compared with Col-0 and irx1. The bri1 and cpd were the mutants with lowest levels of molecular marker mRNA transcripts. The effects of impairment of brassinosteroid signalling also differed between the genes studied, indicating a need for a complete brassinosteroid pathway in UV-B signalling.

Six genes strongly regulated by mercury in Pisum sativum roots.

Suppression subtractive hybridisation was used to isolate heavy metal-induced genes from Pisum sativum roots hydroponically exposed to 5 microM HgCl2 and 10 microM EDTA. Six genes were induced out of which one, PsHMIP6B, was novel. The other genes (PsSAMT, PsI2'H, PsNDA, PsAPSR, PsPOD) had not previously been isolated from pea and sequenced. All six genes were also induced after exposure to 5 microM HgCl2 in the absence of EDTA. The induction pattern was in some cases different for the two Hg species, demonstrating a quicker response to-free Hg2+ than Hg-EDTA. The stress-specificity of the gene regulation was investigated by hydroponically adding 5 microM Cd2+. Most Hg-induced cDNAs were also induced by Cd2+ but to a smaller extent than after Hg exposure. In addition, the gene expression was also probed for tissue specificity, which showed that all six genes were expressed in roots and not in leaves.

Regulation of gene expression by low levels of ultraviolet-B radiation in Pisum sativum: isolation of novel genes by suppression subtractive hybridisation.

Suppression subtractive hybridisation was used to isolate genes differentially regulated by low levels (UV-B(BE,300) 0.13 W m(-2)) of ultraviolet-B radiation (UV-B; 290-320 nm) in Pisum sativum. Six genes were regulated, two of which were novel. The mRNA levels for these two (PsTSDC and PsUOS1) were increased and depressed by UV-B treatment, respectively. Domains in the PsTSDC translation product was similar to TIR (Toll-Interleukin-1 receptor-similar) domains and a NB-ARC domain (nucleotide-binding domain in APAF-1, R gene products and CED-4). The PsUOS1 translation product was similar to an open reading frame in Arabidopsis. Genes encoding embryo-abundant protein (PsEMB) and S-adenosyl-L-methionine synthase (PsSAMS) were induced by UV-B, whereas the transcript levels for genes encoding sucrose transport protein (PsSUT) or ribulose-5-phosphate 3-epimerase (PsR5P3E) were decreased. These regulation patterns are novel, and the PsEMB and PsR5P3E sequences are reported for the first time. The stress-specificity of regulation of these genes were tested by ozone fumigation (100 ppb O(3)). Qualitatively, the similarity of expression after both UV-B and ozone exposure suggests that, for these genes, similar stress-response pathways are in action.