A world of cytochrome P450s

The world we live in is a biosphere influenced by all organisms who inhabit it. It is also an ecology of genes, with some having rather startling effects. The premise put forth in this issue is cytochrome P450 is a significant player in the world around us. Life and the Earth itself would be visibly different and diminished without cytochrome P450s. The contributions to this issue range from evolution on the billion year scale to the colour of roses, from Darwin to Rachel Carson; all as seen through the lens of cytochrome P450.     

The use of thidiazuron in tissue culture

Summary Thidiazuron (N-phenyl-N’-1,2,3-thiadiazol-5-ylurea) was first reported to have cytokinin activity in 1982. Since then, thidiazuron has been used successfully in vitro to induce adventitious shoot formation and to promote axillary shoot proliferation. Thidiazuron is especially effective with recalcitrant woody species. Shoot numbers produced on medium containing thidiazuron are equivalent to or greater than numbers initiated on medium with purine-type cytokinins. Low concentrations of thidiazuron (0.0022 to 0.088 mg/liter) are effective for micropropagation. Prolonged exposure to thidiazuron should be avoided, as this may cause hyperhydricity, abnormal shoot morphology, or problems in rooting. Presented in the Session-in-Depth Novel Plant Growth Regulators at the 1992 World Congress on Cell and Tissue Culture, Washington, D.C., June 20–25, 1992.     

Flow injection assays for NADH and ethanol using photosensitized rose bengal and luminol–copper (II) chemiluminescence system

The online photoreaction of the rose bengal photosensitized luminol–copper (II) chemiluminescence (CL) system was used for the determination of β-nicotinamide adenine dinucleotide (NADH) and ethanol (EtOH) in pharmaceutical formulations combined with a flow injection technique. NADH can significantly enhance the CL emission of the reaction. For EtOH, alcohol dehydrogenase in soluble form was utilized in the presence of nicotinamide adenine dinucleotide resulting in NADH production. The limit of detection (3σ blank, 𝑛 = 3) of 4.0 × 10⁻⁸ and 2.17 × 10⁻⁵ M, and linear range 1.3 × 10⁻⁷ to 2.5 × 10⁻⁵ M (R² = 0.9998, n = 6) and 0.11–2.17 × 10⁻³ M (R² = 0.9996, n = 6) were obtained for NADH and EtOH respectively. The injection rate was 100 h⁻¹ with a relative standard deviation (n = 3) of 1.5–4.8% in the range studied for both analytes. The procedure was satisfactorily applied to pharmaceutical formulations with recoveries in the range 91.6 ± 3.0% to 110 ± 2.0% for NADH and 88 ± 3.0% to 95.4 ± 4.0% for EtOH. The results obtained were very consistent and did not differ considerably from the reported approaches at a 95% confidence limit. The possible mechanism of the CL reaction is also explained briefly.     

Polyamines in Paul's Scarlet rose suspension cultures

Polyamine concentrations have been determined at intervals in suspension cultures of Paul's Scarlet rose cells during a culture period of 2 weeks. The mean concentrations of the putrescine, spermidine and spermine in the cells of the inocula were respectively 73, 70 and 13 nmol/g fresh weight. Putrescine at fitst increased with a peak (160 nmol/g) after 6 h, declined to a minimum (14 nmol/g) after 2–3 days, increased to a second peak (180 nmol/g) after 5–6 days, and then declined slowly to the concentration of the inoculum (taken on day 14). Spermidine rose slowly (×2.6) to a broad peak over 3–6 days (180 nmol/g), then declined slowly to the concentration in the inoculum. Spermine showed a rapid increase to a peak (130 nmol/g) after 2–3 days, and then declined rapidly, reaching the inoculum concentration by day 6. In one experiment the three amines showed a minor peak at day 11. Changes in spermine and RNA contents appeared to be correlated. DNA content reached a peak after that of the RNA (day 3) and did not appear to be correlated with the content of putrescine or the polyamines.     

Hydroxyl Radical Generation Caused by the Reaction of Singlet Oxygen with a Spin Trap,DMPO, Increases Significantly in the Presence of Biological Reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (¹O²) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ¹O² with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (?OH) adduct of DMPO (DMPO-OH) resulting from ¹O²-dependent generation of ?OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its ?OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of ?OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of ?OH from ¹O², and that spin trap-mediated ?OH generation hardly occurs with DEPMPO.     

Modification of Proteinase Inhibitor II in Adzuki Beans during Germination

We investigated the effects of compounds isolated from a methanolic extract of rose hips on melanin biosynthesis in B16 mouse melanoma cells and the possible mechanisms responsible for the inhibition of melanin biosynthesis. We found that, among the isolated compounds, quercetin was a particularly potent melanogenesis inhibitor. To reveal the mechanism for this inhibition, the effects on tyrosinase of B16 mouse melanoma were measured. Quercetin decreased the intracellular tyrosinase activity as well as the tyrosinase activity in a cell culture-free system. We also examined the cellular level of tyrosinase protein and found that quercetin dose-dependently inhibited tyrosinase protein expression. We consider from these results that the inhibition of melanogenesis by quercetin was due to the inhibition of both tyrosinase activity and of the protein expression.     

Production of 2-Phenylethanol in Roses as the Dominant Floral Scent Compound from L-Phenylalanine by Two Key Enzymes,a PLP-Dependent Decarboxylase and a Phenylacetaldehyde Reductase

We investigated the biosynthetic pathway for 2-phenylethanol, the dominant floral scent compound in roses, using enzyme assays. L-[²H₈] Phenylalanine was converted to [²H₈] phenylacetaldehyde and [²H₈]-2-phenylethanol by two enzymes derived from the flower petals of R. ‘Hoh-Jun,’ these being identified as pyridoxal-5′-phosphate-dependent L-aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). The activity of rose petal AADC to yield phenylacetaldehyde was nine times higher toward L-phenylalanine than toward its D-isomer, and this conversion was not inhibited by iproniazid, a specific inhibitor of monoamine oxidase. Under aerobic conditions, rose petal AADC stoichiometrically produced NH₃ together with phenylacetaldehyde during the course of decarboxylation and oxidation, followed by the hydrolysis of L-phenylalanine. Phenylacetaldehyde was subsequently converted to 2-phenylethanol by the action of PAR. PAR showed specificity toward several volatile aldehydes.     

基于过氧化物同工酶分析月季种质资源的亲缘关系及杂种真实性

为了提高种质资源利用率,加速月季育种进程,对27份月季种质及3个杂交组合的8个杂交后代,采用过氧化物酶(POD)同工酶方法分析其亲缘关系并进行杂种真实性鉴定。结果表明:月季种质采用POD同工酶分析具有一定的可行性。酶谱分析中,在相对迁移率为0.264~0.858的位点处共获得7条酶带,其中共有酶带3条,特征酶带4条,表明不同月季种质间遗传多样性丰富,但又存在一定同源性。基于酶带特征进行聚类分析,在相似系数为0.57处,可将27份供试材料分为3个大组。合柱组与月季组材料聚在一个大组中,两个组在形态上的相似性再次得到确认。金樱子与硕苞蔷薇分别聚在两个不同的组中,表明二者之间的亲缘关系较远。供试的古老月季品种被聚在两个不同的大组中,与野生种聚成的一组呈平行关系,表明古老月季在起源上的差异较大,可利用其作为杂交亲本进行广泛杂交以选育具有丰富遗传多样性的杂交后代。根据有无父本特征酶带对杂种后代真实性进行鉴定,初步确定2个杂交组合的6个杂交后代中5个为真实杂种,1个为自交种。该研究结果为进一步开展月季遗传育种奠定了基础。