Sugar cane buds as an efficient explant for plantlet regeneration

An efficient and reproducible protocol for regeneration of plantlets at a high frequency was developed by using sugar cane buds. Disinfected buds were firstly submerged in ethanol sodium hypochlorite solution with 0.1 % polyvinylpyrrolidone, 1.5 % ascorbic acid and 1.75 % citric acid as antioxidants and subsequently treated with solution of agrimicin:captan (1:1). The upper stalk segment was better to obtain bud in vitro culture compared to lower segments. The medium for induction of multiple shoots consisted of Murashige and Skoog basal medium (MS) supplemented with 2 mg dm⁻³ thidiazuron and 1 mg dm⁻³ naphthalene acetic acid. An average of 24 shoots per bud was obtained for cv. Mex 68-P23 within four weeks and 29 shoots for cv. MY 55-14 within six weeks. Indole-3-butyric acid induced more roots in both cultivars compared to the untreated plantlets. Plantlets transferred to soil showed normal growth with up to four axilliary buds in each node. It was concluded that the germplasm obtained through the above mentioned technique generated stalks with more buds in each node which would give farmers more vegetative material for plantations in field with 100 % germination.This research was funded by Fundacion Produce Chiapas A.C. (Mexico).     

N9-substituted derivatives of kinetin: effective anti-senescence agents

The first isolated cytokinin, 6-furfurylaminopurine (kinetin or Kin), was identified almost 55 years ago. Its biological effects on plant cells and tissues include influences on such processes as gene expression, cell cycle, chloroplast development, chlorophyll biosynthesis, stimulation of vascular development, delay of senescence, and mobilization of nutrients. In the present study we prepared a series of eight N9-substituted Kin derivatives, and characterized them with available physicochemical methods such as CI+ mass spectrometry and ¹H NMR spectroscopy. All compounds were tested in three classical cytokinin bioassays: a tobacco callus assay, an Amaranthus assay, and a senescence assay with excised wheat leaves. The ability of the compounds to interact with Arabidopsis cytokinin receptors CRE1/AHK4 and AHK3 was tested in a bacterial receptor assay. Prepared derivatives with certain substitutions of the N9-atom of the purine moiety enhanced the cytokinin activity of the parent compound in the bioassays to a remarkable degree but negatively affected its perception by CRE1/AHK4 and AHK3. The ability of compounds to delay the senescence of excised wheat leaves in both dark and light conditions, was highly correlated with their ability to influence membrane lipid peroxidation, which is a typical symptom of senescence. Our results were corroborated by gene expression profiling of those genes involved in cytokinin metabolism and perception, plant senescence, and the stress response, and suggest that prepared kinetin derivatives might be used as potent anti-senescence agents.     

Cloning of an isozyme of proline 3-hydroxylase and its purification from recombinant Escherichia coli

An isozyme gene of proline 3-hydroxylase was cloned from Streptomyces sp. strain TH1 (Mori H, Shibasaki T, Yano K, Ozaki A, J. Bacteriol. 1997, 179: 5677–5683). The isozyme gene (870 bp) encodes a protein of molecular weight of 33,573. Both 3-hydroxylase genes are identical at 76.2% in amino acid sequence. His-motifs conserved in 2-oxoglutarate-dependent dioxygenases are conserved in both genes. Although characteristics of both recombinant 3-hydroxylases are similar, specific activities to l-proline and proline analogs are different.     

Diversity of 16S rRNA and dioxygenase genes detected in coal‐tar‐contaminated site undergoing active bioremediation

Aims: In order to develop effective bioremediation strategies for polyaromatic hydrocarbons (PAHs) degradation, the composition and metabolic potential of microbial communities need to be better understood, especially in highly PAH contaminated sites in which little information on the cultivation‐independent communities is available. Methods and Results: Coal‐tar‐contaminated soil was collected, which consisted of 122·5 mg g^?1 total extractable PAH compounds. Biodegradation studies with this soil indicated the presence of microbial community that is capable of degrading the model PAH compounds viz naphthalene, phenanthrene and pyrene at 50 ppm each. PCR clone libraries were established from the DNA of the coal‐tar‐contaminated soil, targeting the 16S rRNA to characterize (i) the microbial communities, (ii) partial gene fragment encoding the Rieske iron sulfur center (α‐subunit) common to all PAH dioxygenase enzymes and (iii) β‐subunit of dioxygenase. Phylotypes related to Proteobacteria (Alpha‐, Epsilon‐ and Gammaproteobacteria), Acidobacteria, Actinobacteria, Firmicutes, Gemmatimonadetes and Deinococci were detected in 16S rRNA derived clone libraries. Many of the gene fragment sequences of α‐subunit and β‐subunit of dioxygenase obtained from the respective clone libraries fell into clades that are distinct from the reference dioxygenase gene sequences. Presence of consensus sequence of the Rieske type [2Fe‐2S] cluster binding site suggested that these gene fragments encode for α‐subunit of dioxygenase gene. Conclusions: Sequencing of the cloned libraries representing α‐subunit gene fragments (Rf1) and β‐subunit of dioxygenase showed the presence of hitherto unidentified dioxygenase in coal‐tar‐contaminated soil. Significance and Impact of the Study: The combination of the Rieske primers and bacterial community profiling represents a powerful tool for both assessing bioremediation potential and the exploration of novel dioxygenase genes in a contaminated environment.     

Effect of carbon source on the biotransformation enzymesin Serratia marcescens

A microorganism capable of degrading camphor as the sole source of carbon was isolated from soil. The strain was identified as Serratia marcescens (NCIM 5115). The strain when grown in the peptone–glucose medium showed a doubling time of 2.7 h. This microorganism showed the presence of cytochrome P-450, cytochrome b₅ and the activities of cytochrome c reductase, dichlorophenol indophenol reductase, aminopyrine-N-demethylase and steroid 11--hydroxylase. A significant increase in all activities was observed when cells were incubated for 3h in a medium containing either 0.2% camphor, 1.0% n-hexadecane or 0.1% naphthalene when compared to the peptone–glucose medium.     

2-Hydroxychromene-2-carboxylate isomerase from bacteria that degrade naphthalenesulfonates

2-Hydroxychromene-2-carboxylate isomerase activity was found in cell-free systems from bacteria that degrade naphthalenesulfonates. The enzyme fromPseudomonas testosteroni A3 was activated by incubation with glutathione, dithiothreitol or mercaptoethanol. The highest enzyme activity was found after preincubation of the enzyme with glutathione at alkaline pH-values. A highly purified enzyme preparation converted besides 2-hydroxychromene-2-carboxylate also 2-hydroxybenzo[g]chromene-2-carboxylate (the 2-hydroxychromene-2-carboxylate formed from 1,2-dihydroxyanthracen). The addition of various metal ions or EDTA did not significantly change the catalytic activity of the enzyme. A possible reaction mechanism is proposed.Abbreviations 2,5-DHCCA 2,5-dihydroxychromene-2-carboxylate - 2,6-DHCCA 2,6-dihydroxychromene-2-carboxylate - 1,2-DHN 1,2-dihydroxynaphthalene - GSH glutathione - 2HBCCA 2-hydroxybenzo[g]chromene-2-carboxylate - HBP 2-hydroxybenzalpyruvate - HBPA 2-hydroxybenzalpyruvate aldolase - 2HCCA 2-hydroxychromene-2-carboxylate - 2HCCAI 2-hydroxychromene-2-carboxylate isomerase - 2NS naphthalene-2-sulfonate - R_t retention time     

Cytochrome P450 3A Enzymes Catalyze the O6-Demethylation of Thebaine,a Key Step in Endogenous Mammalian Morphine Biosynthesis

Morphine, first characterized in opium from the poppy Papaver somniferum, is one of the strongest known analgesics. Endogenous morphine has been identified in several mammalian cells and tissues. The synthetic pathway of morphine in the opium poppy has been elucidated. The presence of common intermediates in plants and mammals suggests that biosynthesis occurs through similar pathways (beginning with the amino acid l-tyrosine), and the pathway has been completely delineated in plants. Some of the enzymes in the mammalian pathway have been identified and characterized. Two of the latter steps in the morphine biosynthesis pathway are demethylation of thebaine at the O³- and the O⁶-positions, the latter of which has been difficult to demonstrate. The plant enzymes responsible for both the O³-demethylation and the O⁶-demethylation are members of the Fe^II/α-ketoglutarate-dependent dioxygenase family. Previous studies showed that human cytochrome P450 (P450) 2D6 can catalyze thebaine O³-demethylation. We report that demethylation of thebaine at the O⁶-position is selectively catalyzed by human P450s 3A4 and 3A5, with the latter being more efficient, and rat P450 3A2. Our results do not support O⁶-demethylation of thebaine by an Fe^II/α-ketoglutarate-dependent dioxygenase. In rat brain microsomes, O⁶-demethylation was inhibited by ketoconazole, but not sulfaphenazole, suggesting that P450 3A enzymes are responsible for this activity in the brain. An alternate pathway to morphine, oripavine O⁶-demethylation, was not detected. The major enzymatic steps in mammalian morphine synthesis have now been identified.     

Tryptophan metabolism and disposition in cancer biology and immunotherapy

Tumours utilise tryptophan (Trp) and its metabolites to promote their growth and evade host defences. They recruit Trp through up-regulation of Trp transporters, and up-regulate key enzymes of Trp degradation and down-regulate others. Thus, Trp 2,3-dioxygenase (TDO2), indoleamine 2,3-dioxygenase 1 (IDO1), IDO2, N′-formylkynurenine formamidase (FAMID) and Kyn aminotransferase 1 (KAT1) are all up-regulated in many cancer types, whereas Kyn monooxygenase (KMO), kynureninase (KYNU), 2-amino-3-carboxymuconic acid-6-semialdehyde decarboxylase (ACMSD) and quinolinate phosphoribosyltransferase (QPRT) are up-regulated in a few, but down-regulated in many, cancers. This results in accumulation of the aryl hydrocarbon receptor (AhR) ligand kynurenic acid and in depriving the host of NAD⁺ by blocking its synthesis from quinolinic acid. The host loses more NAD⁺ by up-regulation of the NAD⁺-consuming poly (ADP-ribose) polymerases (PARPs) and the protein acetylaters SIRTs. The nicotinamide arising from PARP and SIRT activation can be recycled in tumours to NAD⁺ by the up-regulated key enzymes of the salvage pathway. Up-regulation of the Trp transporters SLC1A5 and SLC7A5 is associated mostly with that of TDO2 = FAMID > KAT1 > IDO2 > IDO1. Tumours down-regulate enzymes of serotonin synthesis, thereby removing competition for Trp from the serotonin pathway. Strategies for combating tumoral immune escape could involve inhibition of Trp transport into tumours, inhibition of TDO and IDOs, inhibition of FAMID, inhibition of KAT and KYNU, inhibition of NMPRT and NMNAT, inhibition of the AhR, IL-4I1, PARPs and SIRTs, and by decreasing plasma free Trp availability to tumours by albumin infusion or antilipolytic agents and inhibition of glucocorticoid induction of TDO by glucocorticoid antagonism.     

Breathing‐in epigenetic change with vitamin C

Vitamin C is an antioxidant that maintains the activity of iron and α‐ketoglutarate‐dependent dioxygenases. Despite these enzymes being implicated in a wide range of biological pathways, vitamin C is rarely included in common cell culture media. Recent studies show that reprogramming of pluripotent stem cells is enhanced when vitamin C is present, thereby illustrating previous limitations in reprogramming cultures. Here, we summarize understanding of dioxygenase function in reprogramming and epigenetic regulation. The available data suggest a link between dioxygenase function and stem cell differentiation, which is exposed to environmental influence and is relevant for human disease.     

Expression of a nitric oxide degrading enzyme induces a senescence programme in Arabidopsis

Nitric oxide (NO) has been proposed to act as a factor delaying leaf senescence and fruit maturation in plants. Here we show that expression of a NO degrading dioxygenase (NOD) in Arabidopsis thaliana initiates a senescence-like phenotype, an effect that proved to be more pronounced in older than in younger leaves. This senescence phenotype was preceded by a massive switch in gene expression in which photosynthetic genes were down-regulated, whereas many senescence-associated genes (SAGs) and the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene ACS6 involved in ethylene synthesis were up-regulated. External fumigation of NOD plants with NO as well as environmental conditions known to stimulate endogenous NO production attenuated the induced senescence programme. For instance, both high light conditions and nitrate feeding reduced the senescence phenotype and attenuated the down-regulation of photosynthetic genes as well as the up-regulation of SAGs. Treatment of plants with the cytokinin 6-benzylaminopurin (BAP) reduced the down-regulation of photosynthesis, although it had no consistent effect on SAG expression. Metabolic changes during NOD-induced senescence comprehended increases in salicylic acid (SA) levels, accumulation of the phytoalexin camalexin and elevation of leaf gamma-tocopherol contents, all of which occurred during natural senescence in Arabidopsis leaves as well. Moreover, NO fumigation delayed the senescence process induced by darkening individual Arabidopsis Columbia-0 (Col-0) leaves. Our data thus support the notion that NO acts as a negative regulator of leaf senescence.