An Evaluation of the Novel Biological Properties of Diterpenes Isolated from Plectranthus ornatus Codd. In Vitro and In Silico.

Plectranthus ornatus Codd, the genus Plectranthus of the Lamiaceae family, has been used as traditional medicine in Africa, India and Australia. Pharmacological studies show the use of this plant to treat digestive problems. In turn, leaves were used for their antibiotic properties in some regions of Brazil to treat skin infections. The present study examines the anti-inflammatory, antioxidant and cytotoxic effects of the halimane and labdane diterpenes (11R*,13E)-11-acetoxyhalima-5,13-dien-15-oic acid (HAL) and 1α,6ß-diacetoxy-8α,13R*-epoxy-14-labden-11-one (PLEC) and the forskolin-like 1:1 mixture of 1,6-di-O-acetylforskolin and 1,6-di-O-acetyl-9-deoxyforskolin (MRC) isolated from P. ornatus on lung (A549) and leukemia (CCRF-CEM) cancer cell lines, and on normal human retinal pigment epithelial (ARPE-19) cell line in vitro. Additionally, molecular docking and computational approaches were used. ADMET properties were analysed through SwissADME and proTox-II-Prediction. The results indicate that all tested compounds significantly reduced the viability of the cancer cells and demonstrated no cytotoxic effects against the non-neoplastic cell line. The apoptosis indicators showed increased ROS levels for both the tested A549 and CCRF-CEM cancer cell lines after treatment. Furthermore, computational studies found HAL to exhibit moderate antioxidant activity. In addition, selected compounds changed mitochondrial membrane potential (MMP), and increased DNA damage and mitochondrial copy number for the CCRF-CEM cancer cell line; they also demonstrated anti-inflammatory effects on the ARPE-19 normal cell line upon lipopolysaccharide (LPS) treatment, which was associated with the modulation of IL-6, IL-8, TNF-α and GM-CSF genes expression. Docking studies gave indication about the lowest binding energy for 1,6-di-O-acetylforskolin docked into IL-6, TNF-α and GM-CSF, and 1,6-di-O-acetyl-9-deoxyforskolin docked into IL-8. The ADMET studies showed drug-likeness properties for the studied compounds. Thus, halimane and labdane diterpenes isolated from P. ornatus appear to offer biological potential; however, further research is necessary to understand their interactions and beneficial properties.

Diurnal Regulation of the Orexin/Hypocretin System in Mice.

A prominent feature of the hypothalamic neuropeptides orexins/hypocretins is their role in the regulation of sleep-wake behavior. While there is strong evidence for a diurnal (i.e. 24-h) rhythmicity of the expression of prepro-orexin (PPO) and its cleavage products, orexin A and B, it is not known whether orexin receptors are also subject to diurnal regulation. Here we ask whether besides the regulation of PPO the expression of the orexin receptor subtypes OX1R and OX2R varies over 24 hours in the mouse brain. The mRNA levels of PPO, OX1R, and OX2R as well as of various clock genes were analyzed over 24 hours in the hypothalamus, cortex, and adrenal glands of male mice using qPCR. We found a significant diurnal regulation of the mRNA levels of PPO as well as both orexin receptor subtypes in the brain, while no regulation was observed in adrenal glands. While in the cortex the mRNA levels of both OX1R and OX2R showed a significant diurnal regulation, in the hypothalamus, only the OX2R mRNA expression was subject to a diurnal rhythm. The expression of both orexin receptor subtypes significantly correlated with that of clock genes. Remarkably, the expression pattern of OX2R showed a strong and highly significant correlation with that of the clock gene Bmal1 in the cortex and hypothalamus. These results suggest that the rhythmic expression of orexin receptors is linked to clock gene expression and that OX2R may potentially play a role in the timing of sleep-wake behavior.

Antioxidants restore protoporphyrinogen oxidase in variegate porphyria patients.

BACKGROUND: Variegate porphyria (VP) is the result of decreased protoporphyrinogen oxidase (PPOX) activity and results in the accumulation of porphyrins and porphyrin precursors. Our aims were to analyse the basal antioxidant defences and oxidative damage markers and the effects of a diet supplementation with vitamins E and C on the oxidant/antioxidant status and PPOX gene expression in lymphocytes of variegate porphyria (VP) patients. MATERIALS AND METHODS: Twelve women affected by VP and 12 control women participated in a randomized and double-blind crossover study. Each participant took either 50 mg/day vitamin E and 150 mg/day vitamin C or a placebo for 6 months. RESULTS: Lymphocyte PPOX gene expression, together with catalase and glutathione peroxidase activities, was reduced in VP women. No differences were observed in the levels of malondialdehyde and protein carbonyl derivatives. Stimulated lymphocyte H2 O2 production was higher in porphyric women. Supplementation with antioxidant vitamins increased PPOX expression in VP patients. Glutathione reductase (GRd) and superoxide dismutase (SOD) activities were higher in the treatment groups. CONCLUSIONS: Lymphocytes from VP patients show reduced PPOX expression and present a greater susceptibility to producing H2 O2 and impaired H2 O2 detoxifying mechanisms. Supplementation with vitamins E and C restores PPOX expression in VP patients and enhances GRd and SOD activity, suggesting the potential benefits of a diet rich in vitamins E and C in these patients.

Fifty years of porphyria at the University of Cape Town.

The porphyrias are a group of disorders resulting from defective haem biosynthesis. One form, variegate porphyria, is common in South Africa as a result of a founder effect. Over the past 50 years, the University of Cape Town Faculty of Health Sciences has built and maintained an international reputation for excellence in the field of porphyria. The porphyria group is respected for its research and for its accumulated experience in the management of these disorders. Equally important has been the comprehensive and holistic care offered to patients with porphyria, and to their families.

Sampangine inhibits heme biosynthesis in both yeast and human.

The azaoxoaporphine alkaloid sampangine exhibits strong antiproliferation activity in various organisms. Previous studies suggested that it somehow affects heme metabolism and stimulates production of reactive oxygen species (ROS). In this study, we show that inhibition of heme biosynthesis is the primary mechanism of action by sampangine and that increases in the levels of reactive oxygen species are secondary to heme deficiency. We directly demonstrate that sampangine inhibits heme synthesis in the yeast Saccharomyces cerevisiae. It also causes accumulation of uroporphyrinogen and its decarboxylated derivatives, intermediate products of the heme biosynthesis pathway. Our results also suggest that sampangine likely works through an unusual mechanism-by hyperactivating uroporhyrinogen III synthase-to inhibit heme biosynthesis. We also show that the inhibitory effect of sampangine on heme synthesis is conserved in human cells. This study also reveals a surprising essential role for the interaction between the mitochondrial ATP synthase and the electron transport chain.

Subcellular localization and light-regulated expression of protoporphyrinogen IX oxidase and ferrochelatase in Chlamydomonas reinhardtii.

Protoporphyrinogen IX oxidase (PPO) catalyzes the last common step in chlorophyll and heme synthesis, and ferrochelatase (FeC) catalyzes the last step of the heme synthesis pathway. In plants, each of these two enzymes is encoded by two or more genes, and the enzymes have been reported to be located in the chloroplasts or in the mitochondria. We report that in the green alga Chlamydomonas reinhardtii, PPO and FeC are each encoded by a single gene. Phylogenetic analysis indicates that C. reinhardtii PPO and FeC are most closely related to plant counterparts that are located only in chloroplasts. Immunoblotting results suggest that C. reinhardtii PPO and FeC are targeted exclusively to the chloroplast, where they are associated with membranes. These results indicate that cellular needs for heme in this photosynthetic eukaryote can be met by heme that is synthesized in the chloroplast. It is proposed that the multiplicity of genes for PPO and FeC in higher plants could be related to differential expression in differently developing tissues rather than to targeting of different gene products to different organelles. The FeC content is higher in C. reinhardtii cells growing in continuous light than in cells growing in the dark, whereas the content of PPO does not significantly differ in light- and dark-grown cells. In cells synchronized to a light/dark cycle, the level of neither enzyme varied significantly with the phase of the cycle. These results indicate that heme synthesis is not directly regulated by the levels of PPO and FeC in C. reinhardtii.

An Arabidopsis mutant that is resistant to the protoporphyrinogen oxidase inhibitor acifluorfen shows regulatory changes in tetrapyrrole biosynthesis.

Several Arabidopsis mutants of the ecotype Dijon were isolated that show resistance to the herbicide acifluorfen, which inactivates protoporphyrinogen oxidase (PPOX), an enzyme of tetrapyrrole biosynthesis. This enzyme provides protoporphyrin for both Mg chelatase and ferrochelatase at the branchpoint, which leads to chlorophyll and heme, respectively. One of the mutations, aci5-3, displays semidominant inheritance. Heterozygous progeny showed yellow-green leaves, while the homozygous seedlings were white and inviable, but could be rescued by supplementing the medium with sugar. Interestingly, the expression of neither of the two forms of PPOX was altered in the mutant, but the rate of synthesis of 5-aminolevulinate, the precursor of all tetrapyrroles, was drastically reduced. Genetic mapping revealed the mutant locus is closely linked to the ch42 marker, which is itself located in the CHLI-1 gene which codes for one of the three subunits of Mg chelatase. The cs mutant also shows a defect in this gene, and test for allelism with aci5-3 confirmed that the two mutations are allelic. Sequencing of the wild type and aci5-3 alleles of CHLI-1 revealed a single base change (G718A), which results in a D240N substitution in the CHLI-1 protein. In the homozygous aci5-3 mutant no CHLI-1 RNA or protein could be detected. Strikingly, CHLH and CHLI-2 transcripts were also absent. This indicates the existence of a feedback-regulatory mechanism that inactivates the genes encoding certain Mg chelatase subunits. The basis for the semidominant inheritance pattern and the relationship between herbicide resistance and modified gene expression is discussed.

Induction of terminal enzymes for heme biosynthesis during differentiation of mouse erythroleukemia cells.

To examine the induction of terminal enzymes of the heme-biosynthetic pathway during erythroid differentiation, mouse protoporphyrinogen oxidase (PPO) cDNA has been cloned. The deduced amino acid sequence derived from the nucleotide sequence revealed that mouse PPO consists of 477 amino acid residues, without the leader peptide, which is imported into mitochondria. Comparison of the amino terminus of the deduced amino acid sequence of mouse PPO cDNA with that of purified bovine PPO provided conclusive evidence for lack of the leader peptide in the former. The amino acid sequence has 86% and 28% identity with human PPO and Bacillus subtilis HemY, respectively. When mouse erythroleukemia (MEL) cells were induced with dimethylsulfoxide, PPO mRNA was induced within 12 h of treatment, and with further incubation, reached a plateau. mRNAs for coproporphyrinogen oxidase (CPO) and ferrochelatase (FEC) were induced within 12 h, and continued to increase with time up to 48 h. The activities of CPO and FEC markedly increased with time up to 72 h, while PPO activity increased 1.8-fold within 12 h and remained unchanged thereafter. Immunoblot analysis showed that levels of PPO, CPO and FEC paralleled their corresponding activities. The magnitude of PPO induction was less than that of CPO and FEC. Thus, induction of three terminal enzymes of the heme-biosynthetic pathway is an early event in MEL cell differentiation. The concomitant induction may play an important role in producing large amounts of heme during erythroid differentiation.

Effect of the protoporphyrinogen oxidase-inhibiting herbicide fomesafen on liver uroporphyrin and heptacarboxylic porphyrin in two mouse strains.

The effect of the protoporphyrinogen oxidase-inhibiting herbicide fomesafen on liver porphyrin accumulation was studied in long-term high-dose experiments. Fomesafen caused liver accumulation of uroporphyrin and heptacarboxylic porphyrin when fed at 0.25% in the diet to male ICR mice for 5 months (fomesafen-treated mice: 52 nmol uroporphyrin, 21 nmol heptacarboxylic porphyrin/g liver; control mice: traces of uroporphyrin, heptacarboxylic porphyrin not detected). Uroporphyrinogen decarboxylase activity was depressed to about 25% of control values. Iron treatment accelerated the development of this porphyria cutanea tarda-like experimental porphyria both in ICR and C57B1/6J mice. In contrast to other uroporphyrinogen decarboxylase inhibitors, fomesafen treatment did not increase the cytochrome P450IA-related activities and the amount of P450IA2 protein was shown to be significantly decreased by Western immunoblotting. Thus, fomesafen is a unique chemical that inhibits both the oxidation of protoporphyrinogen as well as the conversion of uroporphyrinogen to coproporphyrinogen. However, the accumulation of highly carboxylated porphyrins is evident only after prolonged treatment with high doses of the herbicide.

Investigation of the subcellular location of the tetrapyrrole-biosynthesis enzyme coproporphyrinogen oxidase in higher plants.

The subcellular location of two enzymes in the biosynthetic pathway for protoporphyrin IX, coproporphyrinogen (coprogen) oxidase (EC 1.3.3.3) and protoporphyrinogen (protogen) oxidase (EC 1.3.3.4) has been investigated in etiolated pea (Pisum sativum) leaves and spadices of cuckoo-pint (Arum maculatum). Plant tissue homogenized in isotonic buffer was subjected to subcellular fractionation to prepare mitochondria and plastids essentially free of contamination by other cellular organelles, as determined by marker enzymes. Protogen oxidase activity measured fluorimetrically was reproducibly found in both mitochondria and etioplasts. In contrast, coprogen oxidase could be detected only in etioplasts, using either a coupled fluorimetric assay or a sensitive radiochemical method. The implications of these results for the synthesis of mitochondrial haem in plants is discussed.

Characterization of [3H]acifluorfen binding to purified pea etioplasts, and evidence that protoporphyrinogen oxidase specifically binds acifluorfen.

It is now generally accepted that protoporphyrinogen oxidase is the target-enzyme for diphenyl-ether-type herbicides. Recent studies [Camadro, J-M., Matringe M., Scalla, R. & Labbe, P. (1991) Biochem. J. 277, 17-21] have revealed that in maize, diphenyl ethers competitively inhibit protoporphyrinogen oxidase with respect to its substrate, protoporphyrinogen IX. In this study, we show that, in purified pea etioplast, [3H]acifluorfen specifically binds to a single class of high-affinity binding sites with an apparent dissociation constant of 6.2 +/- 1.3 nM and a maximum density of 29 +/- 5 nmol/g protein. [3H]Acifluorfen binding reaches equilibrium in about 1 min at 30 degrees C. Half dissociation occurs in less than 30 s, indicating that the binding is fully reversible. The specificity of [3H]acifluorfen binding to protoporphyrinogen oxidase is examined. [3H]Acifluorfen binding is inhibited by all the peroxidizing molecules tested. The phthalimide derivative, N-(4-chloro-2-fluoro-5-isopropoxy)phenyl-3,4,5,6-tetra hydrophthalimide, exerts a mixed-competitive inhibition on this binding. The effects of all these molecules on the binding of [3H]acifluorfen are tightly linked to their capacity to inhibit pea etioplast protoporphyrinogen oxidase activity. Furthermore, protoporphyrinogen IX, the substrate of the reaction catalyzed by protoporphyrinogen oxidase, was able to competitively inhibit the binding of [3H]acifluorfen. In contrast, protoporphyrin IX, the product of the reaction, did not inhibit this binding. All these results provide clear evidence that in pea etioplasts, [3H]acifluorfen exclusively binds to protoporphyrinogen oxidase, that the protoporphyrinogen oxidase inhibitors tested so far bind to the same region of the enzyme and that this region overlaps the catalytic site of the enzyme.

Protoporphyrinogen oxidase inhibition by three peroxidizing herbicides: oxadiazon, LS 82-556 and M&B 39279.

Three chemically unrelated peroxidizing molecules, namely oxadiazon [5-(t-butyl)-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2 -one], LS 82-556 [(S)3-N-(methylbenzyl)carbamoyl-5-propionyl-2,6-lutidine] and M&B 39279 [5-amino-4-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)pyrazol], are potent inhibitors of plant, yeast and mouse protoporphyrinogen oxidase.